Fusion proteins and methods for stimulating plant growth, protecting plants from pathogens, and immobilizing Bacillus spores on plant roots

ABSTRACT

The present invention is generally directed to fusion proteins containing a targeting sequence that targets the fusion protein to the exosporium of a  Bacillus cereus  family member. The invention also relates to recombinant  Bacillus cereus  family members expressing such fusion proteins, formulations containing the recombinant  Bacillus cereus  family members expressing the fusion proteins. Methods for stimulating plant growth and for protecting plants from pathogens by applying the recombinant  Bacillus cereus  family members or the formulations to plants or a plant growth medium are also described. The invention also relates to methods for immobilizing spores of a recombinant  Bacillus cereus  family member expressing a fusion protein on plant roots.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/799,262, filed Mar. 15, 2013, the entirety ofwhich is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention generally relates to fusion proteins containing atargeting sequence that targets the fusion protein to the exosporium ofa Bacillus cereus family member. The invention also relates torecombinant Bacillus cereus family members expressing such fusionproteins, formulations containing the recombinant Bacillus cereus familymembers expressing the fusion proteins, and methods for stimulatingplant growth and for protecting plants from pathogens by applying therecombinant Bacillus cereus family members or the formulations to plantsor a plant growth medium. The invention also relates to methods forimmobilizing spores of a recombinant Bacillus cereus family memberexpressing a fusion protein on plant roots.

BACKGROUND OF THE INVENTION

Within the zone surrounding a plant's roots is a region called therhizosphere. In the rhizosphere, bacteria, fungi, and other organismscompete for nutrients and for binding to the root structures of theplant. Both detrimental and beneficial bacteria and fungi can occupy therhizosphere. The bacteria, fungi, and the root system of the plant canall be influenced by the actions of peptides, enzymes, and otherproteins in the rhizosphere. Augmentation of soil or treatment of plantswith certain of these peptides, enzymes, or other proteins would havebeneficial effects on the overall populations of beneficial soilbacteria and fungi, create a healthier overall soil environment forplant growth, improve plant growth, and provide for the protection ofplants against certain bacterial and fungal pathogens. However, previousattempts to introduce peptides, enzymes, and other proteins into soil toinduce such beneficial effects on plants have been hampered by the lowsurvival of enzymes, proteins, and peptides in soil. Additionally, theprevalence of proteases naturally present in the soil leads todegradation of the proteins in the soil. The environment around theroots of a plant (the rhizosphere) is a unique mixture of bacteria,fungi, nutrients, and roots that has different qualities than that ofnative soil. The symbiotic relationship between these organisms isunique, and could be altered for the better with inclusion of exogenousproteins. The high concentration of fungi and bacteria in therhizosphere causes even greater degradation of proteins due toabnormally high levels of proteases and other elements detrimental toproteins in the soil. In addition, enzymes and other proteins introducedinto soil can dissipate away from plant roots quickly.

Thus, there exists a need in the art for a method for effectivelydelivering peptides, enzymes, and other proteins to plant root systemsand for extending the period of time during which such molecules remainactive. Furthermore, there exists a need in the art for a method ofselectively targeting such peptides, enzymes, and proteins to therhizosphere and to plant roots in particular.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a fusion proteincomprising at least one plant growth stimulating protein or peptide anda targeting sequence. The plant growth stimulating protein or peptidecomprises a peptide hormone, a non-hormone peptide, or an enzymeinvolved in the production of a plant growth stimulating compound.

In another aspect, the present invention relates to fusion proteincomprising at least one protein or peptide that protects a plant from apathogen and a targeting sequence.

The present invention is also directed to fusion proteins comprising atleast one root binding protein or peptide and a targeting sequence.

In any of the fusion proteins, the targeting sequence can be:

-   -   (a) a targeting sequence comprising an amino acid sequence        having at least about 43% identity with amino acids 20-35 of SEQ        ID NO: 1, wherein the identity with amino acids 25-35 is at        least about 54%;    -   (b) a targeting sequence comprising amino acids 1-35 of SEQ ID        NO: 1;    -   (c) a targeting sequence comprising amino acids 20-35 of SEQ ID        NO: 1;    -   (d) a targeting sequence comprising SEQ ID NO: 1;    -   (e) an amino acid sequence comprising SEQ ID NO: 2;    -   (f) a targeting sequence comprising amino acids 1-27 of SEQ ID        NO: 3;    -   (g) a targeting sequence comprising amino acids 12-27 of SEQ ID        NO: 3;    -   (h) a targeting sequence comprising SEQ ID NO: 3;    -   (i) an amino acid sequence comprising SEQ ID NO: 4;    -   (j) a targeting sequence comprising amino acids 1-38 of SEQ ID        NO: 5;    -   (k) a targeting sequence comprising amino acids 23-38 of SEQ ID        NO: 5;    -   (l) a targeting sequence comprising SEQ ID NO: 5;    -   (m) an amino acid sequence comprising SEQ ID NO: 6;    -   (n) a targeting sequence comprising amino acids 1-28 of SEQ ID        NO: 7;    -   (o) a targeting sequence comprising amino acids 13-28 of SEQ ID        NO: 7;    -   (p) a targeting sequence comprising SEQ ID NO: 7;    -   (q) an amino acid sequence comprising SEQ ID NO: 8;    -   (r) a targeting sequence comprising amino acids 1-24 of SEQ ID        NO: 9;    -   (s) a targeting sequence comprising amino acids 9-24 of SEQ ID        NO: 9;    -   (t) a targeting sequence comprising SEQ ID NO. 9;    -   (u) an amino acid sequence comprising SEQ ID NO. 10;    -   (v) a targeting sequence comprising amino acids 1-33 of SEQ ID        NO:11;    -   (w) a targeting sequence comprising amino acids 18-33 of SEQ ID        NO: 11;    -   (x) a targeting sequence comprising SEQ ID NO: 11;    -   (y) an amino acid sequence comprising SEQ ID NO: 12;    -   (z) a targeting sequence comprising amino acids 1-33 of SEQ ID        NO: 13;    -   (aa) a targeting sequence comprising amino acids 18-33 of SEQ ID        NO: 13;    -   (ab) a targeting sequence comprising SEQ ID NO:13;    -   (ac) a targeting sequence comprising SEQ ID NO:14;    -   (ad) a targeting sequence comprising amino acids 1-43 of SEQ ID        NO: 15;    -   (ae) a targeting sequence comprising amino acids 28-43 of SEQ ID        NO: 15;    -   (af) a targeting sequence comprising SEQ ID NO:15;    -   (ag) a targeting sequence comprising SEQ ID NO:16;    -   (ah) a targeting sequence comprising amino acids 1-27 of SEQ ID        NO: 17;    -   (ai) a targeting sequence comprising amino acids 12-27 of SEQ ID        NO: 17;    -   (aj) a targeting sequence comprising SEQ ID NO:17;    -   (ak) a targeting sequence comprising SEQ ID NO:18;    -   (al) a targeting sequence comprising amino acids 1-33 of SEQ ID        NO: 19;    -   (am) a targeting sequence comprising amino acids 18-33 of SEQ ID        NO: 19;    -   (an) a targeting sequence comprising SEQ ID NO:19;    -   (ao) a targeting sequence comprising SEQ ID NO:20;    -   (ap) a targeting sequence comprising amino acids 1-33 of SEQ ID        NO: 21;    -   (aq) a targeting sequence comprising amino acids 18-33 of SEQ ID        NO: 21;    -   (ar) a targeting sequence comprising SEQ ID NO:21;    -   (as) a targeting sequence comprising SEQ ID NO:22;    -   (at) a targeting sequence comprising amino acids 1-24 of SEQ ID        NO: 23;    -   (au) a targeting sequence comprising amino acids 9-24 of SEQ ID        NO: 23;    -   (av) a targeting sequence comprising SEQ ID NO:23;    -   (aw) a targeting sequence comprising SEQ ID NO:24;    -   (ax) a targeting sequence comprising amino acids 1-24 of SEQ ID        NO: 25;    -   (ay) a targeting sequence comprising amino acids 9-24 of SEQ ID        NO: 25;    -   (az) a targeting sequence comprising SEQ ID NO:25;    -   (ba) a targeting sequence comprising SEQ ID NO:26;    -   (bb) a targeting sequence comprising amino acids 1-30 of SEQ ID        NO: 27;    -   (bc) a targeting sequence comprising amino acids 15-30 of SEQ ID        NO: 27;    -   (bd) a targeting sequence comprising SEQ ID NO:27;    -   (be) a targeting sequence comprising SEQ ID NO:28;    -   (bf) a targeting sequence comprising amino acids 1-33 of SEQ ID        NO: 29;    -   (bg) a targeting sequence comprising amino acids 18-33 of SEQ ID        NO: 29;    -   (bh) a targeting sequence comprising SEQ ID NO:29;    -   (bi) a targeting sequence comprising SEQ ID NO:30;    -   (bj) a targeting sequence comprising amino acids 1-24 of SEQ ID        NO: 31;    -   (bk) a targeting sequence comprising amino acids 9-24 of SEQ ID        NO: 31;    -   (bl) a targeting sequence comprising SEQ ID NO:31;    -   (bm) a targeting sequence comprising SEQ ID NO:32;    -   (bn) a targeting sequence comprising amino acids 1-15 of SEQ ID        NO: 33;    -   (bo) a targeting sequence comprising SEQ ID NO:33;    -   (bp) a targeting sequence comprising SEQ ID NO:34;    -   (bq) a targeting sequence comprising amino acids 1-16 of SEQ ID        NO: 35;    -   (br) a targeting sequence comprising SEQ ID NO:35; or    -   (bs) a targeting sequence comprising SEQ ID NO:36.

In other aspects, the invention relates to a recombinant Bacillus cereusfamily member that expresses any of the fusion proteins.

In yet other aspects, the invention is directed to formulationscomprising any of the recombinant Bacillus cereus family members and anagriculturally acceptable carrier.

The present invention also relates to a method for stimulating plantgrowth. The method comprises introducing into a plant growth medium anyof the recombinant Bacillus cereus family members expressing a fusionprotein comprising at least one plant growth stimulating protein orpeptide, or any of the formulations comprising the recombinant Bacilluscereus family members expressing a fusion protein comprising at leastone plant growth stimulating protein or peptide. Alternatively, themethod comprises applying to foliage of a plant, a plant seed, or anarea surrounding a plant any of the recombinant Bacillus cereus familymembers expressing a fusion protein comprising at least one plant growthstimulating protein or peptide, or any of the formulations comprisingthe recombinant Bacillus cereus family members expressing a fusionprotein comprising at least one plant growth stimulating protein orpeptide. The plant growth stimulating protein or peptide is physicallyattached to the exosporium of the recombinant Bacillus family member.

Another aspect of the invention is directed to a method for stimulatingplant growth. The method comprises introducing a recombinant Bacilluscereus family member expressing a fusion protein into a plant growthmedium or applying a recombinant Bacillus cereus family memberexpressing a fusion protein to foliage of a plant, a plant seed, or anarea surrounding a plant. The fusion protein comprises at least oneplant growth stimulating protein or peptide and a targeting sequence.The targeting sequence can be any of the targeting sequences listedabove. The plant growth stimulating protein or peptide is physicallyattached to the exosporium of the recombinant Bacillus family member.

The present invention also relates to a method for protecting a plantfrom a pathogen. The method comprises introducing into a plant growthmedium any of the recombinant Bacillus cereus family members expressinga fusion protein comprising at least one protein or peptide thatprotects a plant from a pathogen, or any of the formulations comprisingthe recombinant Bacillus cereus family members expressing a fusionprotein comprising at least one protein or peptide that protects a plantfrom a pathogen. Alternatively, the method comprises applying to foliageof a plant, a plant seed, or an area surrounding a plant any of therecombinant Bacillus cereus family members expressing a fusion proteincomprising at least one protein or peptide that protects a plant from apathogen, or any of the formulations comprising the recombinant Bacilluscereus family members expressing a fusion protein comprising at leastone protein or peptide that protects a plant from a pathogen. Theprotein or peptide that protects a plant from a pathogen is physicallyattached to the exosporium of the recombinant Bacillus family member.

The present invention further relates to a method for immobilizing arecombinant Bacillus cereus family member spore on a root system of aplant. The method comprises introducing into a plant growth medium anyof the recombinant Bacillus cereus family members expressing a fusionprotein comprising at least one root binding protein or peptide, or anyof the formulations comprising the recombinant Bacillus cereus familymembers expressing a fusion protein comprising at least one root bindingprotein or peptide. Alternatively, the method comprises applying tofoliage of a plant, a plant seed, or an area surrounding a plant any ofthe recombinant Bacillus cereus family members expressing a fusionprotein comprising at least one root binding protein or peptide, or anyof the formulations comprising the recombinant Bacillus cereus familymembers expressing a fusion protein comprising at least one root bindingprotein or peptide. The root binding protein or peptide is physicallyattached to the exosporium of the recombinant Bacillus cereus familymember.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows an alignment of the amino acid sequence of theamino-terminal portion of Bacillus anthracis Sterne strain BclA and withthe corresponding region from various exosporium proteins from Bacilluscereus family members.

DEFINITIONS

When the articles “a,” “an,” “one,” “the,” and “said” are used herein,the mean “at least one” or “one or more” unless otherwise indicated.

The terms “comprising,” “including,” and “having” are intended to beinclusive and mean that there may be additional elements other than thelisted elements.

The term “bioactive peptide” refers to any peptide refers to any peptidethat exerts a biological activity. “Bioactive peptides” can begenerated, for example, via the cleavage of a protein or peptide by aprotease or peptidase.

An “enzyme involved in the production of a plant growth stimulatingcompound” includes any enzyme that catalyzes any step in a biologicalsynthesis pathway for a compound that stimulates plant growth. Suchcompounds include, for example, but are not limited to, small moleculeplant hormones such as auxins and cytokinins, bioactive peptides, andsmall plant growth stimulating molecules synthesized by bacteria orfungi in the rhizosphere (e.g., 2,3-butanediol).

The term “fusion protein” as used herein refers to a protein having apolypeptide sequence that comprises sequences derived from two or moreseparate proteins. A fusion protein can be generated by joining togethera nucleic acid molecule that encodes all or part of a first polypeptidewith a nucleic acid molecule that encodes all or part of a secondpolypeptide to create a nucleic acid sequence which, when expressed,yields a single polypeptide having functional properties derived fromeach of the original proteins.

The term “immobilizing a recombinant Bacillus cereus family member sporeon a root system” refers to the binding of a Bacillus cereus familymember spore to a root of a plant, such that the spore is maintained atthe plant's root structure instead of dissipating into the plant growthmedium.

A “plant growth medium” includes any material that is capable ofsupporting the growth of a plant.

A “plant immune system enhancer protein or peptide” as used hereinincludes any protein or peptide that has a beneficial effect on theimmune system of a plant.

The term “plant growth stimulating protein or peptide” as used hereinincludes any protein or peptide that increases plant growth in a plantexposed to the protein or peptide.

A “protein or peptide that protects a plant from a pathogen” as usedherein includes any protein or peptide that makes a plant exposed to theprotein or peptide less susceptible to infection with a pathogen.

The term “root binding protein or peptide” refers to any peptide orprotein capable of specifically or non-specifically binding to a plantroot.

The term “targeting sequence” as used herein refers to a polypeptidesequence that, when present as part of longer polypeptide or a protein,results in the localization of the longer polypeptide or a protein to aspecific subcellular location. The targeting sequences described hereinresult in localization of proteins to the exosporium of a Bacilluscereus family member.

DESCRIPTION OF THE INVENTION

The present invention relates to fusion proteins containing a targetingsequence that targets the fusion protein to the exosporium of a Bacilluscereus family member and: (a) at least one plant growth stimulatingprotein or peptide; (b) at least one protein or peptide that protects aplant from a pathogen; or (c) at least one root binding protein orpeptide. When expressed in Bacillus cereus family member bacteria, thesefusion proteins are targeted to the exosporium layer of the spore andare physically oriented such that the protein or peptide is displayed onthe outside of the spore.

This Bacillus exosporium display (BEMD) system can be used to deliverpeptides, enzymes, and other proteins to plants (e.g., to plant foliageor plant roots) or to a plant growth medium such as soil. Peptides,enzymes, and proteins delivered to the soil or another plant growthmedium in this manner persist and exhibit activity in the soil forextended periods of time. Introduction of recombinant Bacillus cereusfamily member bacteria expressing the fusion proteins described hereininto soil or the rhizosphere of a plant leads to a beneficialenhancement of plant growth in many different soil conditions. The useof the BEMD to create these enzymes allows them to continue to exerttheir beneficial results to the plant and the rhizosphere over the firstyears of a plants life.

Targeting Sequences

For ease of reference, the SEQ ID NOs. for the peptide and proteinsequences referred to herein are listed in Table 1 below.

TABLE 1 Peptide and Protein Sequences Protein or targeting sequence(SEQ ID. NO) Sequence AA 1-41 of Bc1AMSNNNYSNGLNPDESLSASAFDPNLVGPTLPPIPPFTLPTG (B. anthracis Sterne)(SEQ ID NO: 1) Full length Bc1AMSNNNYSNGLNPDESLSASAFDPNLVGPTLPPIPPFTLPTGPTGPFTTG (SEQ ID NO: 2)PTGPTGPTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGPTGPTGPTGPTGFTPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGDTGTTGPTGPTGPTGPTGPTGPTGPTFTGPTGPTGPTGATGLTGPTGPTGPSGLGLPAGLYAFNSGGISLDLGINDPVPFNTVGSQFFTGTAISQLDADTFVISETGFYKITVIANTATASVLGGLTIQVNGVPVPGTGSSLISLGAPFTIVIQAITQITTTPSLVEVIVTGLGLSLALGTSASIIIEKVA AA 1-33 ofMSEKYIILHGTALEPNLIGPTLPPIPPFTFPNG BetA/BAS3290 (B. anthracis Sterne)(SEQ ID NO:3) Full lengthMSEKYIILHGTALEPNLIGPTLPPIPPFTFPNGPTGITGPTGATGFTGIGIT BetA/BAS3290GPTGVTGPTGIGITGPTGATGLGILPVFGTITTDVGIGFSVIVNTNINFTL (SEQ ID NO: 4)PGPVSGTTLNPVDNSIIINTTGVYSVSFSIVFVIQAISSSILNLTINDSIQFAIESRIGGGPGVRATSARTDLLSLNQGDVLRVRIREATGDIIYSNASLV VSKVD Met + AA 2-43 ofMVKVVEGNGGKSKIKSPLNSNFKILSDLVGPTFPPVPTGMTGIT BAS4623(B. anthracis Sterne) (SEQ ID NO: 5) Full length BAS4623VVKVVEGNGGKSKIKSPLNSNFKILSDLVGPTFPPVPTGMTGITGSTG (SEQ ID NO: 6)ATGNTGPTGETGATGSAGITGSTGPTGNTGGTGSTGPTGNTGATGSTGVTGSTGVTGSTGVTGSTGVTGSTGPTGETGGTGSTGVTGSTGATGSTGVTGNTGPTGSTGATGNTGSIGETGGTGSMGPTGETGVTGSTGGTGSTGVTGNTGPTGSTGVTGSTGVTGSTGPTGSTGVTGSTGPTGSTGVTGSTGVTGNMGPTGSTGVTGNTGSTGTTGATGETGPMGSTGATGTTGPTGETGETGETGGTGSTGPTGNTGATGSTGVTGSTGVTGSTGVTGETGPTGSTGATGNTGPTGETGGTGSTGATGSTGVTGNTGPTGSTGVTGNTGATGETGPTGNTGATGNTGPTGETGVTGSTGPTGETGVTGSTGPTGNTGATGETGATGSTGVTGNTGSTGETGPTGSTGPTGSTGATGVTGNTGPTGSTGATGATGSTGPTGSTGTTGNTGVTGDTGPTGATGVSTTATYAFANNTSGSVISVLLGGTNIPLPNNQNIGPGITVSGGNTVFTVANAGNYYIAYTINLTAGLLVSSRITVNGSPLAGTINSPTVATGSFSATIIASLPAGAAVSLQLFGVVALATLSTATPGATLTIIRLS AA 1-34 of Bc1BMKQNDKLWLDKGIIGPENIGPTFPVLPPIHIPTG (B. anthracis Sterne) (SEQ ID NO: 7)Full length Bc1B MKQNDKLWLDKGIIGPENIGPTFPVLPPIHIPTGITGATGATGITGATGP(SEQ ID NO: 8) TGTTGATGATGITGVTGATGITGVTGATGITGVTGATGITGVTGPTGITGATGPTGITGATGPAGITGVTGPTGITGATGPTGTTGVTGPTGDTGLAGATGPTGATGLAGATGPTGDTGATGPTGATGLAGATGPTGATGLTGATGATGATGGGAIIPFASGTTPALLVNAVLANTGTLLGFGFSQPGIAPGVGGTLTILPGVVGDYAFVAPRDGIITSLAGFFSATAALAPLTPVQIQMQIFIAPAASNTFTPVAPPLLLTPALPAIAIGTTATGIQAYNVPVVAGDKILVYVSLTGASPIAAVAGFVSAGLNIV AA 1-30 of BAS1882MDEFLSSAALNPGSVGPTLPPMQPFQFRTG (B. anthracis Sterne) (SEQ ID NO: 9)Full length BAS 1882 MDEFLSSAALNPGSVGPTLPPMQPFQFRTGPTGSTGAKGAIGNTEPYW(SEQ ID NO: 10) HTGPPGIVLLTYDFKSLIISFAFRILPIS AA 1-39 of gene 2280MFDKNEIQKINGILQANALNPNLIGPTLPPIPPFTLPTG (B. weihenstephensis KBAB4)(SEQ ID NO: 11) Full length KBAB4MFDKNEIQKINGILQANALNPNLIGPTLPPIPPFTLPTGPTGVTGPTGVT gene 2280GPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGV (SEQ ID NO: 12)TGPTGVTGPTGVTGPTGVTGPTGVTGPTGETGPTGGTEGCLCDCCVLPMQSVLQQLIGETVILGTIADTPNTPPLFFLFTITSVNDFLVTVTDGTTTFVVNISDVTGVGFLPPGPPITLLPPTDVGCECECRERPIRQLLDAFIGSTVSLLASNGSIAADFSVEQTGLGIVLGTLPINPTTTVRFAISTCKITAVNIT PITMAA 1-39 of gene 3572 MFDKNEMKKTNEVLQANALDPNIIGPTLPPIPPFTLPTG(B. weihenstephensis KBAB4) (SEQ ID NO: 13) Full Length KBAB4MFDKNEMKKTNEVLQANALDPNIIGPTLPPIPPFTLPTGPTGPTGPTGP gene 3572TGPTGPTGPTGPTGPTGPTGPTGPTGPTGLTGPTGPTGLTGPTGLTGPT (SEQ ID NO: 14)GPTGLTGQTGSTGPTGATEGCLCDCCVFPMQEVLRQLVGQTVILATIADAPNVAPRFFLFNITSVNDFLVTVTDPVSNTTFVVNISDVIGVGFSLTVPPLTLLPPADLGCECDCRERPIRELLDTLIGSTVNLLVSNGSIATGFNVEQTALGIVIGTLPIPINPPPPTLFRFAISTCKITAVDITPTPTAT AA 1-49 ofMSRKDKFNRSRMSRKDRFNSPKIKSEISISPDLVGPTFPPIPSFTLPTG Exosporium LeaderPeptide (B. cereus VD200) (SEQ ID NO: 15) Full LengthMSRKDKFNRSRMSRKDRFNSPKIKSEISISPDLVGPTFPPIPSFTLPTGIT Exosporium LeaderGPTFNINFRAEKNVAQSFTPPADIQVSYGNIIFNNGGGYSSVTNTFTAPI PeptideNGIYLFSASIGFNPTLGTTSTLRITIRKNLVSVASQTGTITTGGTPQLEIT (SEQ ID NO: 16)TIIDLLASQTIDIQFSAAESGTLTVGSSNFFSGALLP AA 1-33 ofMNEEYSILHGPALEPNLIGPTLPSIPPFTFPTG Exosporium Leader Peptide(B. cereus VD166) (SEQ ID NO: 17) Full LengthMNEEYSILHGPALEPNLIGPTLPSIPPFTFPTGPTGITGPTGATGFTGIGIT Exosporium LeaderGPTGVTGPTGIGITGPTGATGPTGIGITGPTG Peptide (SEQ ID NO: 18) AA 1-39 ofMKNRDNNRKQNSLSSNFRIPPELIGPTFPPVPTGFTGIG hypothetical protein IKG_04663(B. cereus VD200) (SEQ ID NO: 19) Full LengthMKNRDNNRKQNSLSSNFRIPPELIGPTFPPVPTGFTGIGITGPTGPQGPT hypothetical proteinGPQGPRGLQGPMGEMGPTGPQGVQGIQGSVGPIGATGPEGQQGPQGL IKG_04663, partialRGPQGETGATGPGGVQGLQGPIGPTGATGAQGIQGIQGLQGPIGATGP (SEQ ID NO: 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 1-39 of YVTN β- MSDKHQMKKISEVLQAHALDPNLIGPPLPPITPFTFPTGpropeller protein (B. weihenstephensis KBAB4) (SEQ ID NO: 21)Full length YVTN β- MSDKHQMKKISEVLQAHALDPNLIGPPLPPITPFTFPTGSTGPTGSTGSpropeller protein TGPTGSTGNTGPTGPTGPPVGTNLDTIYVTNDISNNVSAIDGNTNTVLTKBAB4 TIPVGTNPVGVGVNSSTNLIYVVNNGSDNISVINGSTNTVVATIPVGTQ (SEQ ID NO: 22)PFGVGVNPSTNLIYVANRTSNNVSVIKGGTNTVLTTIPVGTNPVGVGVNSSTNLIYVTNEIPNSVSVIKGGTNTVVATIPVGLFPFGVGVNSLTNLIYVVNNSPHNVSVIDGNTNTVLTTISVGTSPVGVGVNLSTNLIYVANEVPNNISVINGNTNTVLTTIPVGTTPFEVGVNSSTNLIYVSNLNSNNVSVINGSANTVIATVPVGSVPRGIGVKP AA 1-30 of MDEFLSFAALNPGSIGPTLPPVPPFQFPTGhypothetical protein bcerkbab4_2363 (B. weihenstephensis KBAB4)(SEQ ID NO: 23) Full lengthMDEFLSFAALNPGSIGPTLPPVPPFQFPTGPTGSTGSTGPTGSTGSTGPT hypothetical proteinGFNLPAGPASITLTSNETTACVSTQGNNTLFFSGQVLVNGSPTPGVVV bcerkbab4_2363SFSFSNPSLAFMVPLAVITNASGNFTAVFLAANGPGTVTVTASLLDSP KBAB4 GTMASVTITIVNCP(SEQ ID NO: 24) AA 1-30 of MDEFLSSTALNPCSIGPTLPPMQPFQFPTGhypothetical protein bcerkbab4_2131 (B. weihenstephensis KBAB4)(SEQ ID NO: 25) Full lengthMDEFLSSTALNPCSIGPTLPPMQPFQFPTGPTGSTGTTGPTGSIGPTGNT hypothetical proteinGLTGNTGPTGITGPTGDTG bcerkbab4 2131 (SEQ ID NO: 26)AA 1-36 of triple helix MKERDRQNSLNSNFRISPNLIGPTFPPVPTGFTGIGrepeat containing collagen (B. weihenstephensis KBAB4) (SEQ ID NO: 27)Full length triple helixMKERDRQNSLNSNFRISPNLIGPTFPPVPTGFTGIGITGPTGPQGPTGPQ repeat-containingGPRGFQGPMGEMGPTGPQGVQGIQGPAGQMGATGPEGQQGPQGLRG collagen KBAB4PQGETGATGPQGVQGLQGPIGPTGATGAQGIQGIQGLQGPIGATGPEG (SEQ ID NO: 28)PQGIQGVQGVPGATGSQGIQGAQGIQGPQGPSGNTGATGVTGQGISGPTGITGPTGITGPSGGPPGPTGATGATGPGGGPSGSTGATGATGNTGVTGSAGVTGNTGSTGSTGETGAQGLQGIQGVQGPIGPTGPEGPQGIQGIPGPTGVTGEQGIQGVQGIQGITGATGDQGPQGIQGAIGPQGITGATGDQGPQGIQGVPGPTGDTGSQGVQGIQGPMGDIGPTGPEGPEGLQGPQGIQGVPGPAGATGPEGPQGIQGIQGPIGVTGPEGPQGIQGIQGIQGITGATGAQGATGVQGVQGNIGATGPEGPQGVQGTQGDIGPTGPMGPQGVQGIQGIQGPTGAQGVQGPQGIQGIQGPTGVTGDTGTTGATGEGTTGATGVTGPSGVTGPSGGPAGPTGPTGPSGPTGLTGPSGGPPGPTGATGVTGGVGDTGATGSTGVTGATGVTGATGATGLQGPQGIQGVQGDIGPTGPQGVQGPQGIQGITGATGDQGPQGIQGPQGIQGPTGPQGIQGGQGPQGIQGATGATGAQGPQGIQGIQGVQGPTGPQGPTGIQGVQGEIGPTGPQGVQGLQGPQGPTGDTGPTGPQGPQGIQGPTGATGATGSQGIQGPTGATGATGSQGIQGPTGATGATGATGATGATGATGATGVTGVSTTATYSFANNTSGSAISVLLGGTNIPLPNNQNIGPGITVSGGNTVFTVTNAGNYYIAYTINITAALLVSSRITVNGSPLAGTINSPAVATGSFNATIISNLAAGSAISLQLFGLLAVATLSTTTPGATLTIIRLS AA 1-39 ofVFDKNEIQKINGILQANALNPNLIGPTLPPIPPFTLPTG hypothetical proteinbmyco0001_21660 (B. mycoides 2048) (SEQ ID NO: 29) Full lengthVFDKNEIQKINGILQANALNPNLIGPTLPPIPPFTLPTGPTGGTGPTGVT hypothetical proteinGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGVTGPTGV bmyco0001_21660TGPTGVTGPTGVTGPTGVTGPTGGTEGCLCDCCVLPMQSVLQQLIGE (SEQ ID NO: 30)TVILGTIADTPNTPPLFFLFTITSVNDFLVTVTDGTTTFVVNISDVTGVGFLPPGPPITLLPPTDVGCECECRERPIRQLLDAFIGSTVSLLASNGSIAADFSVEQTGLGIVLGTLPINPTTTVRFAISTCKITAVNITPITM AA 1-30 ofMDEFLYFAALNPGSIGPTLPPVQPFQFPTG hypothetical protein bmyc0001_22540(B. mycoides 2048) (SEQ ID NO: 31) Full lengthMDEFLYFAALNPGSIGPTLPPVQPFQFPTGPTGSTGATGSTGSTGSTGP hypothetical proteinTGSTGSTGSTGSTGPTGPTGPTGSTGPTGPTGFNLPAGPASITLTSNETT bmyc0001_22540ACVSTQGNNTLFFSGQVLVNGSPTPGVVVSFSFSNPSLAFMVPLAVIT (SEQ ID NO: 32)NASGNFTAVFLAANGPGTVTVTASLLDSPGTMASVTITIVNCP AA 1-21 ofMDSKNIGPTFPPLPSINFPTG hypothetical protein bmyc0001_21510(B. mycoides 2048) (SEQ ID NO: 33) Full lengthMDSKNIGPTFPPLPSINFPTGVTGETGATGETGATGATGETGATGETG hypothetical proteinETGATGATGATGATGETGATGATGATGAAGATGETGATGETGATGE bmyc0001_21510TGATGETGATGVTGETGATGETGAAGETGITGVTGPTGETGATGETG (SEQ ID NO: 34)ATGATGITGATGITGVAGATGETGAAGETGPTGATGAIGAIGATGATGITGVTGATGETGAAGATGITGVTGATGETGAAGATGITGATGITGVAGATGITGPTGIPGTIPTTNLLYFTFSDGEKLIYTNADGIAQYGTTQILSPSEVSYINLFINGILQPQPFYEVTAGQLTLLDDEPPSQGSSIILQFIIIN AA 1-22 of collagenMIGPENIGPTFPILPPIYIPTG triple helix repeat protein (B. thuringiensis35646) (SEQ ID NO: 35) Full length collagenMIGPENIGPTFPILPPIYIPTGETGPTGITGATGETGPTGITGPTGITGATG triple helix repeatETGSTGITGATGETGSTGITGPIGITGATGETGPIGITGATGETGPTGITG proteinSTGITGLTGVTGLTGETGPIGITGPTGITGPTGVTGATGPTGGIGPITTT (SEQ ID NO: 36)NLLYYTFADGEKLIYTDTDGIPQYGTTNILSPSEVSYINLFVNGILQPQPLYEVSTGKLTLLDTQPPSQGSSIILQFIIIN AA = amino acids

Bacillus is a genus of rod-shaped bacteria. The Bacillus cereus familyof bacteria includes the species Bacillus anthracis, Bacillus cereus,Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, andBacillus weihenstephensis. Under stressful environmental conditions,Bacillus cereus family bacteria undergo sporulation and form ovalendospores that can stay dormant for extended periods of time. Theoutermost layer of the endospores is known as the exosporium andcomprises a basal layer surrounded by an external nap of hair-likeprojections. Filaments on the hair-like nap are predominantly formed bythe collagen-like glycoprotein BclA, while the basal layer is comprisedof a number of different proteins. Another collagen-related protein,BclB, is also present in the exosporium and exposed on endospores ofBacillus cereus family members. BclA, the major constituent of thesurface nap, has been shown to be attached to the exosporium with itsamino-terminus (N-terminus) positioned at the basal layer and itscarboxy-terminus (C-terminus) extending outward from the spore.

It was previously discovered that certain sequences from the N-terminalregions of BclA and BclB could be used to target a peptide or protein tothe exosporium of a Bacillus cereus endospore (see U.S. PatentApplication Nos. 2010/0233124 and 2011/0281316, and Thompson et al.,Targeting of the BclA and BclB proteins to the Bacillus anthracis sporesurface, Molecular Microbiology 70(2):421-34 (2008), the entirety ofeach of which is hereby incorporated by reference). It was also foundthat the BetA/BAS3290 protein of Bacillus anthracis localized to theexosporium.

In particular, amino acids 20-35 of BclA from Bacillus anthracis Sternestrain have been found to be sufficient for targeting to the exosporium.A sequence alignment of amino acids 1-41 of BclA (SEQ ID NO: 1) with thecorresponding N-terminal regions of several other Bacillus cereus familyexosporium proteins and Bacillus cereus family proteins having relatedsequences is shown in FIG. 1. The conserved targeting sequence region ofBclA (amino acids 20-35 of SEQ ID NO: 1) is shown in bold, and a morehighly conserved region spanning amino acids 25-35 within the targetingsequence is underlined. SEQ ID NOs. 3, 5, and 7 in FIG. 1 are aminoacids 1-33 of Bacillus anthracis Sterne strain BetA/BAS3290, amethionine followed by amino acids 2-43 of Bacillus anthracis Sternestrain BAS4623, and amino acids 1-34 of Bacillus anthracis Sterne strainBclB, respectively. (For BAS4623, it was found that replacing the valinepresent at position 1 in the native protein with a methionine resultedin better expression.) As can be seen from FIG. 1, each of thesesequences contains a conserved region corresponding to amino acids 20-35of BclA (SEQ ID NO: 1; shown in bold), and a more highly conservedregion corresponding to amino acids 20-35 of BclA (underlined).

Additional proteins from Bacillus cereus family members also contain theconserved targeting region. In particular, in FIG. 1, SEQ ID NO: 9 isamino acids 1-30 of Bacillus anthracis Sterne strain BAS 1882, SEQ IDNO: 11 is amino acids 1-39 of the Bacillus weihenstephensis KBAB4 2280gene product, SEQ ID NO: 13 is amino acids 1-39 of the Bacillusweihenstephensis KBAB4 3572 gene product, SEQ ID NO: 15 is amino acids1-49 of Bacillus cereus VD200 exosporium leader peptide, SEQ ID NO: 17is amino acids 1-33 of Bacillus cereus VD166 exosporium leader peptide,SEQ ID NO: 19 is amino acids 1-39 of Bacillus cereus VD200 hypotheticalprotein IKG_04663, SEQ ID NO: 21 is amino acids 1-39 of Bacillusweihenstephensis KBAB4 YVTN β-propeller protein, SEQ ID NO: 23 is aminoacids 1-30 of Bacillus weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2363, SEQ ID NO: 25 is amino acids 1-30 of Bacillusweihenstephensis KBAB4 hypothetical protein bcerkbab4_2131, SEQ ID NO:27 is amino acids 1-36 of Bacillus weihenstephensis KBAB4 triple helixrepeat containing collagen, SEQ ID NO: 29 is amino acids 1-39 ofBacillus mycoides 2048 hypothetical protein bmyco0001_21660, SEQ ID NO:31 is amino acids 1-30 of Bacillus mycoides 2048 hypothetical proteinbmyc0001_22540, SEQ ID NO: 33 is amino acids 1-21 of Bacillus mycoides2048 hypothetical protein bmyc0001_21510, and SEQ ID NO: 35 is aminoacids 1-22 of Bacillus thuringiensis 35646 collagen triple helix repeatprotein. As shown in FIG. 1, each of the N-terminal regions of theseproteins contains a region that is conserved with amino acids 20-35 ofBclA (SEQ ID NO: 1), and a more highly conserved region corresponding toamino acids 25-35 of BclA.

In the fusion proteins of the present invention, any portion of BclAwhich includes amino acids 20-35, including full length BclA, can beused as the targeting sequence in the present invention.

Alternatively, any portion of BetA/BAS3290, BAS4623, BclB, BAS1882, theKBAB4 2280 gene product, the KBAB4 3572 gene product, B. cereus VD200exosporium leader peptide, B. cereus VD166 exosporium leader peptide, B.cereus VD200 hypothetical protein IKG_04663, B. weihenstephensis KBAB4YVTN β-propeller protein, B. weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2363, B. weihenstephensis KBAB4 hypothetical proteinbcerkbab4_2131, B. weihenstephensis KBAB4 triple helix repeat containingcollagen, B. mycoides 2048 hypothetical protein bmyco0001_21660, B.mycoides 2048 hypothetical protein bmyc0001_22540, B. mycoides 2048hypothetical protein bmyc0001_21510, or B. thuringiensis 35646 collagentriple helix repeat protein which includes the amino acids correspondingto amino acids 20-35 of BclA can serve as the targeting sequence. As canbe seen from FIG. 1, amino acids 12-27 of BetA/BAS3290, amino acids23-38 of BAS4623, amino acids 13-28 of BclB, amino acids 9-24 ofBAS1882, amino acids 18-33 of KBAB4 2280 gene product, amino acids 18-33of KBAB4 3572 gene product, amino acids 28-43 of B. cereus VD200exosporium leader peptide, amino acids 12-27 of B. cereus VD166exosporium leader peptide, amino acids 18-33 of B. cereus VD200hypothetical protein IKG_04663, amino acids 18-33 B. weihenstephensisKBAB4 YVTN β-propeller protein, amino acids 9-24 of B. weihenstephensisKBAB4 hypothetical protein bcerkbab4_2363, amino acids 9-24 of B.weihenstephensis KBAB4 hypothetical protein bcerkbab4_2131, amino acids15-30 of B. weihenstephensis KBAB4 triple helix repeat containingcollagen, amino acids 18-33 of B. mycoides 2048 hypothetical proteinbmyco0001_21660, amino acids 9-24 of B. mycoides 2048 hypotheticalprotein bmyc0001_22540, amino acids 1-15 of B. mycoides 2048hypothetical protein bmyc0001_21510, and amino acids 1-16 of B.thuringiensis 35646 collagen triple helix repeat protein correspond toamino acids 20-35 of BclA. Thus, any portion of these proteins thatincludes the above-listed corresponding amino acids can serve as thetargeting sequence.

Furthermore, any amino acid sequence comprising amino acids 20-35 ofBclA, or any of the above-listed corresponding amino acids can serve asthe targeting sequence.

Thus, the targeting sequence can comprise amino acids 1-35 of SEQ ID NO:1, amino acids 20-35 of SEQ ID NO: 1, or SEQ ID NO: 1. Alternatively,the targeting sequence consists of amino acids 1-35 of SEQ ID NO: 1,amino acids 20-35 of SEQ ID NO: 1, or SEQ ID NO: 1. Alternatively, thetargeting sequence can comprise full length BclA (SEQ ID NO: 2).

The targeting sequence can also comprise amino acids 1-27 of SEQ ID NO:3, amino acids 12-27 of SEQ ID NO: 3, SEQ ID NO: 3, or full lengthBetA/BAS3290 (SEQ ID NO: 4).

The targeting sequence can also comprise amino acids 1-38 of SEQ ID NO:5, amino acids 23-38 of SEQ ID NO: 5, SEQ ID NO: 5, or full lengthBAS4623 (SEQ ID NO: 6).

Alternatively, the targeting sequence can comprise amino acids 1-28 ofSEQ ID NO: 7, amino acids 13-28 of SEQ ID NO: 7, SEQ ID NO: 7, or fulllength BclB (SEQ ID NO:8).

The targeting sequence can also comprise amino acids 1-24 of SEQ ID NO:9, amino acids 9-24 of SEQ ID NO: 9, SEQ ID NO. 9, or full lengthBAS1882 (SEQ ID NO. 10).

The targeting sequence can also comprise amino acids 1-33 of SEQ IDNO:11, amino acids 18-33 of SEQ ID NO: 11, SEQ ID NO: 11, or the fulllength KBAB4 2280 gene product (SEQ ID NO: 12).

The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO:13, amino acids 18-33 of SEQ ID NO: 13, SEQ ID NO:13, or the full lengthKBAB4 3572 gene product (SEQ ID NO:14).

Alternatively, the targeting sequence can comprise amino acids 1-43 ofSEQ ID NO: 15, amino acids 28-43 of SEQ ID NO: 15, SEQ ID NO:15, or fulllength B. cereus VD200 exosporium leader peptide (SEQ ID NO:16).

The targeting sequence can also comprise amino acids 1-27 of SEQ ID NO:17, amino acids 12-27 of SEQ ID NO: 17, SEQ ID NO:17, or full-length B.cereus VD166 exosporium leader peptide (SEQ ID NO:18).

The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO:19, amino acids 18-33 of SEQ ID NO: 19, SEQ ID NO:19, or full length B.cereus VD200 hypothetical protein IKG_04663 (SEQ ID NO:20).

Alternatively, the targeting sequence comprises amino acids 1-33 of SEQID NO: 21, amino acids 18-33 of SEQ ID NO: 21, SEQ ID NO:21, or fulllength B. weihenstephensis KBAB4 YVTN β-propeller protein (SEQ IDNO:22).

The targeting sequence can also comprise amino acids 1-24 of SEQ ID NO:23, amino acids 9-24 of SEQ ID NO: 23, SEQ ID NO:23, or full length B.weihenstephensis KBAB4 hypothetical protein bcerkbab4_2363 (SEQ IDNO:24).

The targeting sequence comprise amino acids 1-24 of SEQ ID NO: 25, aminoacids 9-24 of SEQ ID NO: 25, SEQ ID NO:25, or full length B.weihenstephensis KBAB4 hypothetical protein bcerkbab4_2131 (SEQ IDNO:26).

Alternatively, the targeting sequence comprises amino acids 1-30 of SEQID NO: 27, amino acids 15-30 of SEQ ID NO: 27, SEQ ID NO:27, or fulllength B. weihenstephensis KBAB4 triple helix repeat containing collagen(SEQ ID NO:28).

The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO:29, amino acids 18-33 of SEQ ID NO: 29, SEQ ID NO:29, or full length B.mycoides 2048 hypothetical protein bmyco0001_21660 (SEQ ID NO:30).

The targeting sequence can also comprise amino acids 1-24 of SEQ ID NO:31, amino acids 9-24 of SEQ ID NO: 31, SEQ ID NO:31, or full length B.mycoides 2048 hypothetical protein bmyc0001_22540 (SEQ ID NO:32).

Alternatively, the targeting sequence comprises amino acids 1-15 of SEQID NO: 33, SEQ ID NO:33, or full length B. mycoides 2048 hypotheticalprotein bmyc0001_21510 (SEQ ID NO:34).

The targeting sequence can also comprise amino acids 1-16 of SEQ ID NO:35, SEQ ID NO:35, or full length B. thuringiensis 35646 collagen triplehelix repeat protein (SEQ ID NO:36).

In addition, it can readily be seen from the sequence alignment in FIG.1 that while amino acids 20-35 of BclA are conserved, and amino acids25-35 are more conserved, some degree of variation can occur in thisregion without affecting the ability of the targeting sequence to targeta protein to the exosporium. FIG. 1 lists the percent identity of eachof corresponding amino acids of each sequence to amino acids 20-35 ofBclA (“20-35% Identity”) and to amino acids 25-35 of BclA (“25-35%Identity”). Thus, for example, as compared to amino acids 20-35 of BclA,the corresponding amino acids of BetA/BAS3290 are about 81.3% identical,the corresponding amino acids of BAS4623 are about 50.0% identical, thecorresponding amino acids of BclB are about 43.8% identical, thecorresponding amino acids of BAS1882 are about 62.5% identical, thecorresponding amino acids of the KBAB4 2280 gene product are about 81.3%identical, and the corresponding amino acids of the KBAB4 3572 geneproduct are about 81.3% identical. The sequence identities over thisregion for the remaining sequences are listed in FIG. 1.

With respect to amino acids 25-35 of BclA, the corresponding amino acidsof BetA/BAS3290 are about 90.9% identical, the corresponding amino acidsof BAS4623 are about 72.7% identical, the corresponding amino acids ofBclB are about 54.5% identical, the corresponding amino acids of BAS1882are about 72.7% identical, the corresponding amino acids of the KBAB42280 gene product are about 90.9% identical, and the corresponding aminoacids of the KBAB4 3572 gene product are about 81.8% identical. Thesequence identities over this region for the remaining sequences arelisted in FIG. 1.

Thus, the targeting sequence can comprise an amino acid sequence havingat least about 43% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 54%.Alternatively, the targeting sequence consists of an amino acid sequencehaving at least about 43% identity with amino acids 20-35 of SEQ ID NO.1, wherein the identity with amino acids 25-35 is at least about 54%.

The targeting sequence can also comprise an amino acid sequence havingat least about 50% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 63%.Alternatively the targeting sequence consists of an amino acid sequencehaving at least about 50% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 63%.

The targeting sequence can also comprise an amino acid sequence havingat least about 50% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 72%.Alternatively, the targeting sequence consists of an amino acid sequencehaving at least about 50% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 72%.

The targeting sequence can also comprises an amino acid sequence havingat least about 56% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 63%.Alternatively, the targeting sequence consists of an amino acid sequencehaving at least about 56% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 63%.

Alternatively, the targeting sequence can comprise an amino sequencehaving at least about 62% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 72%.The targeting sequence can also consist of an amino acid sequence havingat least about 62% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 of SEQ ID NO:1 is at leastabout 72%.

The targeting sequence can also comprises an amino sequence having atleast about 75% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 72%.Alternatively, the targeting sequence consists of an amino acid sequencehaving at least about 75% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 of SEQ ID NO:1 is atleast about 72%.

The targeting sequence can also comprise an amino sequence having atleast about 75% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 81%.Alternatively, the targeting sequence consists of an amino acid sequencehaving at least about 75% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 of SEQ ID NO:1 is atleast about 81%.

The targeting sequence can also comprise an amino acid sequence havingat least about 81% identity with amino acids 20-35 of SEQ ID NO:1,wherein the identity with amino acids 25-35 is at least about 81%.Alternatively, the targeting sequence consists of an amino acid sequencehaving at least about 81% identity with amino acids 20-35 of SEQ IDNO:1, wherein the identity with amino acids 25-35 is at least about 81%.

The targeting sequence can comprise an amino acid sequence having atleast about 81% identity with amino acids 20-35 of SEQ ID NO: 1, whereinthe identity with amino acids 25-35 is at least about 90%.Alternatively, the targeting sequence consists of an amino acid sequencehaving at least about 81% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 90%.

The skilled person will recognize that variants of the above sequencescan also be used as targeting sequences, so long as the targetingsequence comprises amino acids 20-35 of BclA, the corresponding aminoacids of BetA/BAS3290, BAS4263, BclB, BAS1882, the KBAB4 2280 geneproduct, or the KBAB 3572 gene product, or a sequence comprising any ofthe above noted sequence identities to amino acids 20-35 and 25-35 ofBclA is present.

In any of the above targeting sequences, the targeting sequence cancomprise the amino acid sequence GXT at its carboxy terminus, wherein Xis any amino acid.

Fusion Proteins

The present invention relates to fusion proteins comprising a targetingsequence and at least one plant growth stimulating protein or peptide,wherein the plant growth stimulating protein or peptide comprises apeptide hormone, a non-hormone peptide, or an enzyme involved in theproduction of a plant growth stimulating compound.

The present invention also relates to fusion proteins comprising atargeting sequence and at least one protein or peptide that protects aplant from a pathogen.

In addition, the present invention relates to fusion proteins comprisinga targeting sequence and at least one root binding protein or peptide.

In any of the fusion proteins described herein, the targeting sequencecan be any of the targeting sequences described above in the precedingsection.

The fusion protein can be made using standard cloning and molecularbiology methods known in the art. For example, a gene encoding a proteinor peptide (e.g., a gene encoding a plant growth stimulating protein orpeptide) can be amplified by polymerase chain reaction (PCR) and ligatedto DNA coding for any of the above-described targeting sequences to forma DNA molecule that encodes the fusion protein. The DNA moleculeencoding the fusion protein can be cloned into any suitable vector, forexample a plasmid vector. The vector suitably comprises a multiplecloning site into which the DNA molecule encoding the fusion protein canbe easily inserted. The vector also suitably contains a selectablemarker, such as an antibiotic resistance gene, such that bacteriatransformed with the vector can be readily identified and isolated.Where the vector is a plasmid, the plasmid suitably also comprises anorigin of replication. The DNA encoding the fusion protein is suitablyunder the control of a promoter which will cause expression of thefusion protein on the exosporium of a B. cereus family member endospore(e.g., a native bclA promoter from a B. cereus family member).

The fusion protein can also comprise additional polypeptide sequencesthat are not part of the targeting sequence or the plant growthstimulating protein or peptide, the protein or peptide that protects aplant from a pathogen, or the root binding protein or peptide. Forexample, the fusion protein can include tags or markers to facilitatepurification or visualization of the fusion protein (e.g., apolyhistidine tag or a fluorescent protein such as GFP or YFP).

Plant Growth Stimulating Proteins and Peptides

As noted above, the present invention relates to fusion proteinscomprising a targeting sequence and at least one plant growthstimulating protein or peptide, wherein the plant growth stimulatingprotein or peptide comprises a peptide hormone, a non-hormone peptide,or an enzyme involved in the production of a plant growth stimulatingcompound.

For example, where the plant growth stimulating protein or peptidecomprises a peptide hormone, the peptide hormone can comprise aphytosulfokine (e.g., phytosulfokine-α), clavata 3 (CLV3), systemin,ZmlGF, or a SCR/SP11.

Where the plant growth stimulating protein or peptide comprises anon-hormone peptide, the non-hormone peptide can comprise a RKN 16D10,Hg-Syv46, an eNOD40 peptide, RHPP, or kunitz trypsin inhibitor.

The plant growth stimulating protein or peptide can comprise an enzymeinvolved in the production of a plant growth stimulating compound. Theenzyme involved in the production of a plant growth stimulating compoundcan be any enzyme that catalyzes any step in a biological synthesispathway for a compound that stimulates plant growth.

The plant growth stimulating compound can comprise a compound producedby bacteria or fungi in the rhizosphere, e.g., 2,3-butanediol.

Alternatively, the plant growth stimulating compound can comprise aplant growth hormone, e.g., a cytokinin or an auxin.

Where the plant growth stimulating compound comprises a cytokinin, thecytokinin can comprise kinetin, cis-zeatin, trans-zeatin,6-benzylaminopurine, dihydroxyzeatin, N6-(D2-isopentenyl)adenine,ribosylzeatin, N6-(D2-isopentenyl)adenosine,2-methylthio-cis-ribosylzeatin, cis-ribosylzeatin, trans-ribosylzeatin,2-methylthio-trans-ribosylzeatin, ribosylzeatin-5-monosphosphate,N6-methylaminopurine, N6-dimethylaminopurine, 2′-deoxyzeatin riboside,4-hydroxy-3-methyl-trans-2-butenylaminopurine, ortho-topolin,meta-topolin, benzyladenine, ortho-methyltopolin, meta-methyltopolin, ora combination thereof.

Where the plant growth stimulating compound comprises an auxin, theauxin can comprise indole-3-acetic acid, indole-3-pyruvic acid,indole-3-acetaldoxime, indole-3-acetamide, indole-3-acetonitrile,indole-3-ethanol, indole-3-pyruvate, indole-3-acetaldoxime,indole-3-butyric acid, a phenylacetic acid, or 4-chloroindole-3-aceticacid, or a combination thereof.

The enzyme involved in the production of a plant growth stimulatingcompound can comprise an acetoin reductase, an indole-3-acetamidehydrolase, a tryptophan monoxygenase, an acetolactate synthetase, anα-acetolactate decarboxylase, a pyruvate decarboxylase, a diacetylreductase, a butanediol dehydrogenase, an aminotransferase (e.g.,tryptophan aminotransferase), a tryptophan decarboxylase, an amineoxidase, an indole-3-pyruvate decarboxylase, an indole-3-acetaldehydedehydrogenase, a tryptophan side chain oxidase, a nitrile hydrolase, anitrilase, a peptidase, a protease, an adenosine phosphateisopentenyltransferase, a phosphatase, an adenosine kinase, an adeninephosphoribosyltransferase, CYP735A, a 5′ ribonucleotidephosphohydrolase, an adenosine nucleosidase, a zeatin cis-transisomerase, a zeatin O-glucosyltransferase, a β-glucosidase, acis-hydroxylase, a CK cis-hydroxylase, a CK N-glucosyltransferase, a2,5-ribonucleotide phosphohydrolase, an adenosine nucleosidase, a purinenucleoside phosphorylase, or a zeatin reductase.

Where the enzyme comprises a protease or peptidase, the protease orpeptidase can be a protease or peptidase that cleaves proteins orpeptides to create a bioactive peptide. The bioactive peptide can be anypeptide that exerts a biological activity.

Examples of bioactive peptides include RKN 16D10 and RHPP.

The protease or peptidase that cleaves proteins or peptides to create abioactive peptide can comprise subtilisin, an acid protease, an alkalineprotease, a proteinase, an endopeptidase, an exopeptidase, thermolysin,papain, pepsin, trypsin, pronase, a carboxylase, a serine protease, aglutamic protease, an aspartate protease, a cysteine protease, athreonine protease, or a metalloprotease.

The protease or peptidase can cleave proteins in a protein-rich meal(e.g., soybean meal or yeast extract).

Proteins and Peptides that Protects Plants from Pathogens

The present invention relates to fusion proteins comprising a targetingsequence and at least one protein or peptide that protects a plant froma pathogen.

For example, the protein or peptide that protects a plant from apathogen can comprise a plant immune system enhancer protein or peptide.The plant immune system enhancer protein or peptide can be any proteinor peptide that has a beneficial effect on the immune system of a plant.Suitable plant immune system enhancer proteins and peptides includeharpins, α-elastins, β-elastins, cryptogeins, flagellin proteins, andflagellin peptides.

Alternatively, the protein or peptide that protects a plant from apathogen can be a protein or peptide that has antibacterial activity,antifungal activity, or both antibacterial and antifungal activity.Examples of such proteins and peptides include bacteriocins, lysozymes,siderophores, avidins, streptavidins, conalbumins, albumins, orlactoferrins.

The protein that protects a plant from a pathogen can comprise anenzyme. Suitable enzymes include proteases and lactonases. The proteasesand lactonases can be specific for a bacterial signaling molecule (e.g.,a bacterial lactone homoserine signaling molecule).

Where the enzyme is a lactonase, the lactonase can comprise1,4-lactonase, 2-pyrone-4,6-dicarboxylate lactonase, 3-oxoadipateenol-lactonase, actinomycin lactonase, deoxylimonate A-ring-lactonase,gluconolactonase L-rhamnono-1,4-lactonase, limonin-D-ring-lactonase,steroid-lactonase, triacetate-lactonase, or xylono-1,4-lactonase.

The enzyme can also be an enzyme that is specific for a cellularcomponent of a bacterium or fungus. For example, the enzyme can comprisea β-1,3-glucanase, a β-1,4-glucanase, a β-1,6-glucanase, a chitosinase,a chitinase, a chitosinase-like enzyme, a lyticase, a peptidase, aproteinase, a protease (e.g., an alkaline protease, an acid protease, ora neutral protease), a mutanolysin, a stapholysin, or a lysozyme.

For any of the above fusion proteins comprising a protein or peptidethat protects a plant from a pathogen, the pathogen can be a bacterialpathogen or a fungal pathogen. For example, the pathogen can comprise anα-class Proteobacterium, a β-class Proteobacterium, a γ-classProteobacterium, or a combination thereof. Particular bacterialpathogens include Agrobacterium tumefaciens, Pantoea stewartii, Erwiniacarotovora, Ralstonia solanacearum, Pseudomonas syringae, Pseudomonasaeruginosa, Xanthomonas campestris, and combinations thereof.

Other pathogens include Acarosporina microspora, Aceria guerreronis,Achlya conspicua, Achlya klebsiana, Achlysiella williamsi,Acholeplasmataceae, Acidovorax avenae, Acremonium strictum, Acrocalymmamedicaginis, Acrodontium simplex, Acrophialophora fusispora, Acrosporiumtingitaninum, Aecidium, Aecidium aechmantherae, Aecidium amaryllidis,Aecidium breyniae, Aecidium campanulastri, Aecidium cannabis, Aecidiumcantensis, Aecidium caspicum, Aecidium foeniculi, Agrobacteriumtumefaciens, Albonectria rigidiuscula, Albugo bliti, Albugo candida,Albugo ipomoeae-panduratae, Albugo laibachii, Albugo occidentalis,Albugo tragopogonis, Alternaria, Alternaria alternate, Alternariabrassicae, Alternaria brassicicola, Alternaria carthami, Alternariacinerariae, Alternaria citri, Alternaria dauci, Alternaria dianthi,Alternaria dianthicola, Alternaria euphorbiicola, Alternaria helianthi,Alternaria helianthicola, Alternaria japonica, Alternaria leucanthemi,Alternaria limicola, Alternaria linicola, Alternaria mali, Alternariapadwickii, Alternaria panax, Alternaria radicina, Alternaria raphani,Alternaria saponariae, Alternaria senecionis, Alternaria solani,Alternaria tenuissima, Alternaria triticina, Alternaria zinniae,Amazonia, Amphobotrys ricini, Anguillosporella vermiformis, Anguina(genus), Anguina agrostis, Anguina amsinckiae, Anguina australis,Anguina balsamophila, Anguina funesta, Anguina graminis, Anguinaspermophaga, Anguina tritici, Anisogramma anomala, Anthostomellapullulans, Antrodia albida, Antrodia serialiformis, Antrodia serialis,Aphanomyces cladogamus, Aphanomyces cochlioides, Aphanomyces euteiches,Aphanomyces euteiches f.sp. pisi, Aphanomyces raphani, Aphelenchoides,Aphelenchoides arachidis, Aphelenchoides besseyi, Aphelenchoidesfragariae, Aphelenchoides parietinus, Aphelenchoides ritzemabosi,Aphelenchus avenae, Apiognomonia errabunda, Apiognomonia veneta,Apiospora montagnei, Appendiculella, Armillaria affinis, Armillariaapalosclera, Armillaria camerunensis, Armillaria duplicate, Armillariafellea, Armillaria fumosa, Armillaria fuscipes, Armillaria griseomellea,Armillaria heimii, Armillaria mellea, Armillaria melleorubens,Armillaria montagnei, Armillaria omnituens, Armillaria pallidula,Armillaria paulensis, Armillaria pelliculata, Armillaria procera,Armillaria puiggarii, Armillaria singular, Armillaria socialis,Armillaria solidipes, Armillaria tabescens, Armillaria tigrensis,Armillaria umbrinobrunnea, Armillaria viridiflava, Armillaria yungensis,Arthrocladiella, Arthuriomyces peckianus, Ascochyta asparagine,Ascochyta bohemica, Ascochyta caricae, Ascochyta doronici, Ascochytafabae f.sp. lentis, Ascochyta graminea, Ascochyta hordei, Ascochytahumuli, Ascochyta pisi, Ascochyta prasadii, Ascochyta sorghi, Ascochytaspinaciae, Ascochyta tarda, Ascochyta tritici, Ascospora ruborum, Ashbyagossypii, Aspergillus aculeatus, Aspergillus fischerianus, Aspergillusniger, Asperisporium caricae, Asperisporium minutulum, Asteridiella,Asteridiella perseae, Asteroma caryae, Asteroma coryli, Asteromainconspicuum, Athelia arachnoidea, Athelia Aurantiporus fissilis,Belonolaimus, Belonolaimus gracilis, Belonolaimus longicaudatus,Beniowskia sphaeroidea, Bionectria ochroleuca, Bipolaris, Bipolariscactivora, Bipolaris cookie, Bipolaris incurvate, Bipolaris sacchari,Biscogniauxia capnodes var. capnodes, Biscogniauxia marginate,Biscogniauxia nummularia, Bjerkandera adusta, Blakeslea trispora,Blumeria graminis, Botryodiplodia oncidii, Botryodiplodia ulmicola,Botryosphaeria cocogena, Botryosphaeria corticola, Botryosphaeriadisrupta, Botryosphaeria dothidea, Botryosphaeria marconii,Botryosphaeria obtuse, Botryosphaeria quercuum, Botryosphaeria rhodina,Botryosphaeria ribis, Botryosphaeria stevensii, Botryosporium pulchrum,Botryotinia, Botryotinia fuckeliana, Botrytis anthophila, Botrytiscinerea, Botrytis fabae, Bremia lactucae, Brenneria salicis, Briosiaampelophaga, Bulbomicrosphaera, Burkholderia andropogonis, Burkholderiacaryophylli, Burkholderia glumae, Cadophora malorum, Caespitotheca,Calonectria indusiata, Calonectria kyotensis, Calonectriaquinqueseptata, Calvatia versispora, Camarosporium pistaciae,Camarotella acrocomiae, Camarotella costaricensis, CandidatusLiberibacter, Capitorostrum cocoes, Capnodium footii, Capnodiummangiferum, Capnodium ramosum, Capnodium theae, Caulimoviridae,Cephaleuros virescens, Cephalosporium gramineum, Ceratobasidium cereal,Ceratobasidium cornigerum, Ceratobasidium noxium, Ceratobasidiumramicola, Ceratobasidium setariae, Ceratobasidium stevensii,Ceratocystis adiposa, Ceratocystis coerulescens, Ceratocystis fimbriata,Ceratocystis moniliformis, Ceratocystis paradoxa, Ceratocystis pilifera,Ceratocystis pluriannulata, Ceratorhiza hydrophile, Ceratospermopsis,Cercoseptoria ocellata, Cercospora, Cercospora angreci, Cercospora apii,Cercospora apii f.sp. clerodendri, Cercospora apiicola, Cercosporaarachidicola, Cercospora asparagi, Cercospora atrofiliformis, Cercosporabeticola, Cercospora brachypus, Cercospora brassicicola, Cercosporabrunkii, Cercospora cannabis, Cercospora cantuariensis, Cercosporacapsici, Cercospora carotae, Cercospora corylina, Cercospora fragariae,Cercospora fuchsiae, Cercospora fusca, Cercospora fusimaculans,Cercospora gerberae, Cercospora halstedii, Cercospora handelii,Cercospora hayi, Cercospora hydrangea, Cercospora kikuchii, Cercosporalentis, Cercospora liquidambaris, Cercospora longipes, Cercosporalongissima, Cercospora mamaonis, Cercospora mangiferae, Cercosporamedicaginis, Cercospora melongenae, Cercospora minima, Cercosporaminuta, Cercospora nicotianae, Cercospora odontoglossi, Cercosporapapaya, Cercospora penniseti, Cercospora pisa-sativae, Cercosporaplatanicola, Cercospora puderii, Cercospora pulcherrima, Cercosporarhapidicola, Cercospora rosicola, Cercospora rubrotincta, Cercosporasojina, Cercospora solani, Cercospora solani-tuberosi, Cercosporasorghi, Cercospora theae, Cercospora tuberculans, Cercospora vexans,Cercospora vicosae, Cercospora zeae-maydis, Cercospora zebrina,Cercospora zonata, Cercosporella rubi, Cereal cyst nematode, Ceriporiaspissa, Ceriporia xylostromatoides, Cerrena unicolor, Ceuthospora lauri,Choanephora, Choanephora cucurbitarum, Choanephora infundibulifera,Chondrostereum purpureum, Chrysomyxa ledi var. rhododendri, Chrysomyxaledicola, Chrysomyxa piperiana, Chrysomyxa roanensis, Cladosporium,Cladosporium arthropodii, Cladosporium caryigenum, Cladosporiumcladosporioides, Cladosporium cladosporioides f.sp. pisicola,Cladosporium cucumerinum, Cladosporium herbarum, Cladosporium musae,Cladosporium oncobae, Clavibacter michiganensis, Claviceps fusiformis,Claviceps purpurea, Claviceps sorghi, Claviceps zizaniae, Climacodonpulcherrimus, Climacodon septentrionalis, Clitocybe parasitica,Clonostachys rosea f. rosea, Clypeoporthe iliau, Cochliobolus,Cochliobolus carbonum, Cochliobolus cymbopogonis, Cochliobolushawaiiensis, Cochliobolus heterostrophus, Cochliobolus lunatus,Cochliobolus miyabeanus, Cochliobolus ravenelii, Cochliobolus sativus,Cochliobolus setariae, Cochliobolus spicifer, Cochliobolus stenospilus,Cochliobolus tuberculatus, Cochliobolus victoriae, Coleosporiumhelianthi, Coleosporium ipomoeae, Coleosporium madiae, Coleosporiumpacificum, Coleosporium tussilaginis, Colletotrichum acutatum,Colletotrichum arachidis, Colletotrichum capsici, Colletotrichumcereale, Colletotrichum crassipes, Colletotrichum dematium,Colletotrichum dematium f. spinaciae, Colletotrichum derridis,Colletotrichum destructivum, Colletotrichum fragariae, Colletotrichumgossypii, Colletotrichum higginsianum, Colletotrichum kahawae,Colletotrichum lindemuthianum, Colletotrichum lini, Colletotrichummangenotii, Colletotrichum musae, Colletotrichum nigrum, Colletotrichumorbiculare, Colletotrichum pisi, Colletotrichum sublineolum,Colletotrichum trichellum, Colletotrichum trifolii, Colletotrichumtruncatum, Coniella castaneicola, Coniella diplodiella, Coniellafragariae, Coniothecium chomatosporum, Coniothyrium celtidis-australis,Coniothyrium henriquesii, Coniothyrium rosarum, Coniothyriumwernsdorffiae, Coprinopsis psychromorbida, Cordana johnstonii, Cordanamusae, Coriolopsis floccose, Coriolopsis gallica, Corticium invisum,Corticium penicillatum, Corticium theae, Coryneopsis rubi, Corynesporacassiicola, Coryneum rhododendri, Crinipellis sarmentosa, Cronartiumribicola, Cryphonectriaceae, Cryptocline cyclaminis, Cryptomeliola,Cryptoporus volvatus, Cryptosporella umbrina, Cryptosporiopsistarraconensis, Cryptosporium minimum, Curvularia caricae-papayae,Curvularia penniseti, Curvularia senegalensis, Curvularia trifolii,Cylindrocarpon candidum, Cylindrocarpon ianthothele var. ianthothele,Cylindrocarpon magnusianum, Cylindrocarpon musae, Cylindrocladiellacamelliae, Cylindrocladiella parva, Cylindrocladium clavatum,Cylindrocladium lanceolatum, Cylindrocladium peruvianum, Cylindrocladiumpteridis, Cylindrosporium cannabinum, Cylindrosporium juglandis,Cylindrosporium rubi, Cymadothea trifolii, Cytospora, Cytosporapalmarum, Cytospora personata, Cytospora platani, Cytospora sacchari,Cytospora sacculus, Cytospora terebinthi, Cytosporina ludibunda,Dactuliophora elongata, Daedaleopsis confragosa, Dasineura urticae,Datronia scutellata, Davidiella carinthiaca, Davidiella dianthi,Davidiella tassiana, Deightoniella papuana, Deightoniella torulosa,Dendrophoma marconii, Dendrophora erumpens, Denticularia mangiferae,Dermea pseudotsugae, Diaporthaceae, Diaporthe, Diaporthe arctii,Diaporthe citri, Diaporthe dulcamarae, Diaporthe eres, Diaporthehelianthi, Diaporthe lagunensis, Diaporthe lokoyae, Diaporthe melonis,Diaporthe orthoceras, Diaporthe perniciosa, Diaporthe phaseolorum,Diaporthe phaseolorum var. caulivora, Diaporthe phaseolorum var.phaseolorum, Diaporthe phaseolorum var. sojae, Diaporthe rudis,Diaporthe tanakae, Diaporthe toxica, Dibotryon morbosum, Dicarpelladryina, Didymella bryoniae, Didymella fabae, Didymella lycopersici,Didymosphaeria arachidicola, Didymosphaeria taiwanensis, Dilophosphoraalopecuri, Dimeriella sacchari, Diplocarpon earlianum, Diplocarpon mali,Diplocarpon mespili, Diplocarpon rosae, Diplodia laelio-cattleyae,Diplodia manihoti, Diplodia paraphysaria, Diplodia theae-sinensis,Discosia artocreas, Guignardia fulvida, Discostroma corticola,Distocercospora, Distocercospora livistonae, Ditylenchus, Ditylenchusafricanus, Ditylenchus angustus, Ditylenchus destructor, Ditylenchusdipsaci, Dolichodorus heterocephalus, Dothideomycetes, Dothiorellaaromatic, Dothiorella dominicana, Dothiorella gregaria, Dothiorellaulmi, Drechslera avenacea, Drechslera campanulata, Drechsleradematioidea, Drechslera gigantea, Drechslera glycines, Drechsleramusae-sapientium, Drechslera teres f. maculate, Drechslerawirreganensis, Durandiella pseudotsugae, Eballistra lineata, Eballistraoryzae, Eballistraceae, Echinodontium tinctorium, Ectendomeliola,Elsinoë ampelina, Elsinoë australis, Elsinoë batatas, Elsinoëbrasiliensis, Elsinoë fawcettii, Elsinoë leucospila, Elsinoë mangiferae,Elsinoë pini, Elsinoë randii, Elsinoë rosarum, Elsinoë sacchari, Elsinoëtheae, Elsinoë veneta, Endomeliola, Endothia radicalis, Endothiellagyrosa, Entoleuca mammata, Entorrhizomycetes, Entyloma ageratinae,Entyloma dahlia, Entyloma ellisii, Epicoccum nigrum, Ergot, Erwinia,Erwinia chrysanthemi, Erwinia psidii, Erysiphaceae, Erysiphales,Erysiphe, Erysiphe alphitoides, Erysiphe betae, Erysiphebrunneopunctata, Erysiphe cichoracearum, Erysiphe cruciferarum, Erysipheflexuosa, Erysiphe graminis f. sp. avenae, Erysiphe graminis f.sp.tritici, Erysiphe heraclei, Erysiphe pisi, Eutypella parasitica,Eutypella scoparia, Exobasidium burtii, Exobasidium reticulatum,Exobasidium vaccinii var. japonicum, Exobasidium vaccinii-uliginosi,Exobasidium vexans, Exophiala, Flavescence donée, Fomes fasciatus, Fomeslamaënsis, Fomes meliae, Fomitopsis cajanderi, Fomitopsis palustris,Fomitopsis rosea, Fomitopsis spraguei, Fomitopsis supina, Formaspecialis, Frommeella tormentillae, Fusarium, Fusarium affine, Fusariumarthrosporioides, Fusarium circinatum, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium incarnatum, Fusarium solani,Fusarium merismoides, Fusarium oxysporum f.sp. albedinis, Fusariumoxysporum f.sp. asparagi, Fusarium oxysporum f.sp. batatas, Fusariumoxysporum f.sp. betae, Fusarium oxysporum f.sp. cannabis, Fusariumoxysporum f.sp. citri, Fusarium oxysporum f.sp. coffea, Fusariumoxysporum f.sp. cubense, Fusarium oxysporum f.sp. cyclaminis, Fusariumoxysporum f.sp. dianthi, Fusarium oxysporum f.sp. lentis, Fusariumoxysporum f.sp. lini, Fusarium oxysporum f.sp. lycopersici, Fusariumoxysporum f.sp. medicaginis, Fusarium oxysporum f.sp. pisi, Fusariumoxysporum f.sp. radicis-lycopersici, Fusarium pallidoroseum, Fusariumproliferatum, Fusarium redolens, Fusarium sacchari, Fusarium solanif.sp. pisi, Fusarium sporotrichioides, Fusarium subglutinans, Fusariumsulphureum, Fuscoporia torulosa, Fusicladium pisicola, Fusicoccumaesculi, Fusicoccum amygdali, Gaeumannomyces graminis var tritici,Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var.graminis, Galactomyces candidum, Ganoderma brownii, Ganoderma lobatum,Ganoderma orbiforme, Ganoderma philippii, Ganoderma tornatum, Ganodermazonatum, Geastrumia polystigmatis, Georgefischeriaceae,Georgefischeriales, Geosmithia morbida, Geotrichum, Geotrichum candidum,Geotrichum candidum var. citri-aurantii, Geotrichum klebahnii,Gibberella, Gibberella acuminata, Gibberella avenacea, Gibberellabaccata, Gibberella cyanogena, Gibberella fujikuroi, Gibberellafujikuroi var. subglutinans, Gibberella intricans, Gibberella pulicaris,Gibberella stilboides, Gibberella xylarioides, Gibberella zeae,Gibellina cerealis, Gilbertella persicaria, Gjaerumiaceae, Gliocladiumvermoeseni, Globodera pallida, Globodera rostochiensis, Globoderatabacum, Gloeocercospora sorghi, Gloeocystidiellum porosum, Gloeophyllummexicanum, Gloeophyllum trabeum, Gloeoporus dichrous, Gloeosporiumcattleyae, Gloeosporium theae-sinensis, Glomerella cingulate, Glomerellaglycines, Glomerella graminicola, Glomerella tucumanensis, Gnomoniacaryae, Gnomonia comari, Gnomonia dispora, Gnomonia iliau, Gnomonialeptostyla, Gnomonia nerviseda, Gnomonia rubi, Golovinomycescichoracearum var. latisporus, Granulobasidium vellereum, Graphiolaphoenicis, Graphium rigidum, Graphium rubrum, Graphyllium pentamerum,Grovesinia pyramidalis, Guignardia bidwellii f. muscadinii, Guignardiacamelliae, Guignardia citricarpa, Guignardia mangiferae, Guignardiamusae, Guignardia philoprina, Gummosis, Gymnoconia nitens, Gymnopusdryophilus, Gymnosporangium clavipes, Gymnosporangium sabinae,Gymnosporangium globosum, Gymnosporangium juniperi-virginianae,Gymnosporangium kernianum, Gymnosporangium nelsonii, Gymnosporangiumyamadae, Haematonectria haematococca, Hansenula subpelliculosa,Hapalosphaeria deformans, Haplobasidion musae, Haustorium,Helicobasidium compactum, Helicobasidium longisporum, Helicobasidiumpurpureum, Helicoma muelleri, Helicotylenchus, Helicotylenchusdihystera, Helicotylenchus multicinctus, Helminthosporium cookei,Helminthosporium papulosum, Helminthosporium solani, Helotiales,Hemicriconemoides kanayaensis, Hemicriconemoides mangiferae,Hemicycliophora arenaria, Hemlock woolly adelgid, Hendersoniacreberrima, Hendersonia theicola, Hericium coralloides, Heterobasidionannosum, Heterodera, Heterodera amygdali, Heterodera arenaria,Heterodera aucklandica, Heterodera avenae, Heterodera bergeniae,Heterodera bifenestra, Heterodera cacti, Heterodera cajani, Heteroderacanadensis, Heterodera cardiolata, Heterodera carotae, Heteroderaciceri, Heterodera cruciferae, Heterodera delvii, Heterodera elachista,Heterodera filipjevi, Heterodera gambiensis, Heterodera goettingiana,Heterodera hordecalis, Heterodera humuli, Heterodera latipons,Heterodera medicaginis, Heterodera oryzae, Heterodera oryzicola,Heterodera rosii, Heterodera sacchari, Heterodera schachtii, Heteroderatabacum, Heterodera trifolii, Heteroderidae, Hexagonia hydnoides,Hirschmanniella oryzae, Hoplalaimus galeatus, Hoplolaimidae, Hoplolaimuscolumbus, Hoplolaimus indicus, Hoplolaimus magnistylus, Hoplolaimuspararobustus, Hoplolaimus seinhorsti, Hoplolaimus uniformis,Huanglongbing, Hyaloperonospora, Hyaloperonospora arabidopsidis,Hyaloperonospora brassicae, Hyaloperonospora parasitica, Hymenulaaffinis, Hyphodermella corrugata, Hyphodontia aspera, Hyphodontiasambuci, Hypochnus, Hypoxylon tinctor, Idriella lunata, Inonotusarizonicus, Inonotus cuticularis, Inonotus dryophilus, Inonotushispidus, Inonotus ludovicianus, Inonotus munzii, Inonotus tamaricis,Irenopsis, Irpex destruens, Irpex lacteus, Isariopsis clavispora,Johncouchia mangiferae, Kabatiella caulivora, Kabatiella lini, Karnalbunt, Khuskia oryzae, Kretzschmaria deusta, Kretzschmaria zonata,Kuehneola uredinis, Kutilakesa pironii, Labrella coryli, Laeticorticiumroseum, Laetiporus baudonii, Lagenocystis radicicola, Laricifomesofficinalis, Lasiodiplodia theobromae, Leandria momordicae, Leifsoniaxyli xyli, Lentinus tigrinus, Lenzites betulina, Lenzites elegans,Lepteutypa cupressi, Leptodontidium elatius var. elatius, Leptographiummicrosporum, Leptosphaeria acuta, Leptosphaeria cannabina, Leptosphaeriaconiothyrium, Leptosphaeria libanotis, Leptosphaeria lindquistii,Leptosphaeria maculans, Leptosphaeria musarum, Leptosphaeria pratensis,Leptosphaeria sacchari, Leptosphaeria woroninii, Leptosphaerulinacrassiasca, Leptosphaerulina trifolii, Leptothyrium nervisedum,Leptotrochila medicaginis, Leucocytospora leucostoma, Leucostomaauerswaldii, Leucostoma kunzei, Leucostoma persoonii, Leveillulacompositarum f. helianthi, Leveillula leguminosarum f. lentis,Leveillula taurica, Ligniera pilorum, Limacinula tenuis, Linochoragraminis, Longidorus africanus, Longidorus maximus, Longidorus sylphus,Lopharia crassa, Lophodermium, Lophodermium aucupariae, Lophodermiumschweinitzii, Lophodermium seditiosum, Macrophoma mangiferae, Macrophomatheicola, Macrophomina phaseolina, Macrosporium cocos, Magnaporthe,Magnaporthe grisea, Magnaporthe salvinii, Mamianiella coryli,Marasmiellus cocophilus, Marasmiellus inoderma, Marasmiellus scandens,Marasmiellus stenophyllus, Marasmius crinis-equi, Marasmius sacchari,Marasmius semiustus, Marasmius stenophyllus, Marasmius tenuissimus,Massarina walkeri, Mauginiella scaettae, Melampsora, Melampsora linivar. lini, Melampsora medusae, Melampsora occidentalis, Melanconiscarthusiana, Melanconium juglandinum, Meliola, Meliola mangiferae,Meliolaceae, Meloidogyne acronea, Meloidogyne arenaria, Meloidogyneartiellia, Meloidogyne brevicauda, Meloidogyne chitwoodi, Meloidogyneenterolobii, Meloidogyne fruglia, Meloidogyne gajuscus, Meloidogyneincognita, Meloidogyne javanica, Meloidogyne naasi, Meloidogynepartityla, Meloidogyne thamesi, Meripilus giganteus, Merliniusbrevidens, Meruliopsis ambigua, Mesocriconema xenoplax, Microascusbrevicaulis, Microbotryum violaceum, Microdochium bolleyi, Microdochiumdimerum, Microdochium panattonianum, Microdochium phragmitis,Microsphaera, Microsphaera coryli, Microsphaera diffusa, Microsphaeraellisii, Microsphaera euphorbiae, Microsphaera hommae, Microsphaerapenicillata, Microsphaera penicillata var. vaccinii, Microsphaeravaccinii, Microsphaera verruculosa, Microstroma juglandis, Moesziomycesbullatus, Monilinia azaleae, Monilinia fructicola, Monilinia fructigena,Monilinia laxa, Monilinia mali, Moniliophthora perniciosa,Moniliophthora roreri, Monilochaetes infuscans, Monochaetia coryli,Monochaetia mali, Monographella albescens, Monographella cucumerina,Monographella nivalis var. neglecta, Monographella nivalis var. nivalis,Mononegavirales, Monosporascus cannonballus, Monosporascus eutypoides,Monostichella coryli, Mucor circinelloides, Mucor hiemalis, Mucorhiemalis f. silvaticus, Mucor mucedo, Mucor paronychius, Mucorpiriformis, Mucor racemosus, Mycena citricolor, Mycena maculate,Mycocentrospora acerina, Mycoleptodiscus terrestris, Mycosphaerellaangulata, Mycosphaerella arachidis, Mycosphaerella areola,Mycosphaerella berkeleyi, Mycosphaerella bolleana, Mycosphaerellabrassicicola, Mycosphaerella caricae, Mycosphaerella caryigena,Mycosphaerella cerasella, Mycosphaerella citri, Mycosphaerellacoffeicola, Mycosphaerella confusa, Mycosphaerella cruenta,Mycosphaerella dendroides, Mycosphaerella eumusae, Mycosphaerellafragariae, Mycosphaerella gossypina, Mycosphaerella graminicola,Mycosphaerella henningsii, Mycosphaerella horii, Mycosphaerellajuglandis, Mycosphaerella lageniformis, Mycosphaerella linicola,Mycosphaerella louisianae, Mycosphaerella musae, Mycosphaerellamusicola, Mycosphaerella palmicola, Mycosphaerella pinodes,Mycosphaerella pistaciarum, Mycosphaerella pistacina, Mycosphaerellaplatanifolia, Mycosphaerella polymorpha, Mycosphaerella pomi,Mycosphaerella punctiformis, Mycosphaerella pyri, Didymella rabiei,Mycosphaerella recutita, Mycosphaerella rosicola, Mycosphaerella rubi,Mycosphaerella stigmina-platani, Mycosphaerella striatiformans,Mycovellosiella concors, Mycovellosiella fulva, Mycovellosiella koepkei,Mycovellosiella vaginae, Myriogenospora aciculispora, Myrotheciumroridum, Myrothecium verrucaria, Nacobbus aberrans, Nacobbus dorsalis,Naevala perexigua, Naohidemyces vaccinii, Nectria, Nectria cinnabarina,Nectria coccinea, Nectria ditissima, ectria foliicola, Nectria mammoideavar. rubi, Nectria mauritiicola, Nectria peziza, Nectria pseudotrichia,Nectria radicicola, Nectria ramulariae, Nectriella pironii, Nemaniadiffusa, Nemania serpens var. serpens, Nematospora coryli, Neocosmosporavasinfecta, Neodeightonia phoenicum, Neoerysiphe, Neofabraeamalicorticis, Neofabraea perennans, Neofusicoccum mangiferae, Neonectriagalligena, Oidiopsis gossypii, Oidium (genus), Oidium arachidis, Oidiumcaricae-papayae, Oidium indicum, Oidium mangiferae, Oidium manihotis,Oidium tingitaninum, Olpidium brassicae, Omphalia tralucida,Oncobasidium theobromae, Onnia tomentosa, Ophiobolus anguillides,Ophiobolus cannabinus, Ophioirenina, Ophiostoma ulmi, Ophiostomawageneri, Ovulariopsis papayae, Ovulinia azaleae, Ovulitis azaleae,Oxyporus corticola, Oxyporus latemarginatus, Oxyporus populinus,Oxyporus similis, Ozonium texanum var. parasiticum, Paecilomyces fulvus,Paralongidorus maximus, Paratrichodorus christiei, Paratrichodorusminor, Paratylenchus curvitatus, Paratylenchus elachistus, Paratylenchushamatus, Paratylenchus macrophallus, Paratylenchus microdorus,Paratylenchus projectus, Paratylenchus tenuicaudatus, Pathovar, Pauahia,Peach latent mosaic viroid, Pectobacterium carotovorum, Peltasterfructicola, Penicillium aurantiogriseum, Penicillium digitatum,Penicillium expansum, Penicillium funiculosum, Penicillium glabrum,Penicillium italicum, Penicillium purpurogenum, Penicillium ulaiense,Peniophora, Peniophora albobadia, Peniophora cinerea, Peniophoraquercina, Peniophora sacrata, Perenniporia fraxinea, Perenniporiafraxinophila, Perenniporia medulla-panis, Perenniporia subacida,Periconia circinate, Periconiella cocoes, Peridesmium califormicum,Peronosclerospora miscanthi, Peronosclerospora sacchari,Peronosclerospora sorghi, Peronospora, Peronospora anemones, Peronosporaantirrhini, Peronospora arborescens, Peronospora conglomerata,Peronospora destructor, Peronospora dianthi, Peronospora dianthicola,Peronospora farinosa, Peronospora farinosa f.sp. betae, Peronosporahyoscyami f.sp. tabacina, Peronospora manshurica, Peronosporapotentillae, Peronospora sparsa, Peronospora trifoliorum, Peronosporavalerianellae, Peronospora viciae, Pestalosphaeria concentrica,Pestalotia longiseta, Pestalotia longisetula, Pestalotia rhododendri,Pestalotiopsis, Pestalotiopsis adusta, Pestalotiopsis arachidis,Pestalotiopsis disseminata, Pestalotiopsis guepini, Pestalotiopsisleprogena, Pestalotiopsis longiseta, Pestalotiopsis mangiferae,Pestalotiopsis palmarum, Pestalotiopsis sydowiana, Pestalotiopsis theae,Pestalotiopsis versicolor, Phacidiopycnis padwickii, Phacidiuminfestans, Phaeochoropsis mucosa, Phaeocytostroma iliau, Phaeocytostromasacchari, Phaeoisariopsis bataticola, Phaeolus schweinitzii,Phaeoramularia angolensis, Phaeoramularia dissiliens, Phaeoramulariaheterospora, Phaeoramularia manihotis, Phaeoseptoria musae,Phaeosphaerella mangiferae, Phaeosphaerella theae, Phaeosphaeriaavenaria f.sp. avenaria, Phaeosphaeria avenaria f.sp. triticae,Phaeosphaeria herpotrichoides, Phaeosphaeria microscopica, Phaeosphaerianodorum, Phaeosphaeriopsis obtusispora, Phaeotrichoconis crotalariae,Phakopsora gossypii, Phakopsora pachyrhizi, Phanerochaete allantospora,Phanerochaete arizonica, Phanerochaete avellanea, Phanerochaete burtii,Phanerochaete carnosa, Phanerochaete chrysorhizon, Phanerochaeteradicata, Phanerochaete salmonicolor, Phanerochaete tuberculate,Phanerochaete velutina, Phellinus ferreus, Phellinus gilvus, Phellinusigniarius, Phellinus pini, Phellinus pomaceus, Phellinus weirii,Phialophora asteris, Phialophora cinerescens, Phialophora gregata,Phialophora tracheiphila, Phloeospora multimaculans, Pholiotavariicystis, Phoma, Phoma caricae-papayae, Phoma clematidina, Phomacostaricensis, Phoma cucurbitacearum, Phoma destructiva, Phoma draconis,Phoma eupyrena, Phoma exigua, Phoma exigua var. exigua, Phoma exiguavar. foveata, Phoma exigua var. linicola, Phoma glomerate, Phomaglycinicola, Phoma herbarum, Phoma insidiosa, Phoma medicaginis, Phomamicrospora, Phoma nebulosa, Phoma oncidii-sphacelati, Phoma pinodella,Phoma scabra, Phoma sclerotioides, Phoma strasseri, Phoma tracheiphila,Phomopsis arnoldiae, Phomopsis asparagi, Phomopsis asparagicola,Phomopsis azadirachtae, Phomopsis cannabina, Phomopsis caricae-papayae,Phomopsis coffeae, Phomopsis elaeagni, Phomopsis ganjae, Phomopsisjavanica, Phomopsis lokoyae, Phomopsis mangiferae, Phomopsis obscurans,Phomopsis perseae, Phomopsis prunorum, Phomopsis scabra, Phomopsissclerotioides, Phomopsis tanakae, Phomopsis theae, Photoassimilate,Phragmidium, Phragmidium mucronatum, Phragmidium rosae-pimpinellifoliae,Phragmidium rubi-idaei, Phragmidium violaceum, Phyllachora cannabis,Phyllachora graminis var. graminis, Phyllachora gratissima, Phyllachoramusicola, Phyllachora pomigena, Phyllachora sacchari, Phyllactinia,Phyllactinia angulata, Phyllactinia guttata, Phyllody, Phyllosticta,Phyllosticta alliariaefoliae, Phyllosticta anacardiacearum, Phyllostictaarachidis-hypogaeae, Phyllosticta batatas, Phyllosticta capitalensis,Phyllosticta caricae-papayae, Phyllosticta carpogena, Phyllostictacircumscissa, Phyllosticta coffeicola, Phyllosticta concentrica,Phyllosticta coryli, Phyllosticta cucurbitacearum, Phyllostictacyclaminella, Phyllosticta erratica, Phyllosticta hawaiiensis,Phyllosticta lentisci, Phyllosticta manihotis, Phyllosticta micropuncta,Phyllosticta mortonii, Phyllosticta nicotianae, Phyllosticta palmetto,Phyllosticta penicillariae, Phyllosticta perseae, Phyllosticta platani,Phyllosticta pseudocapsici, Phyllosticta sojaecola, Phyllostictasolitaria, Phyllosticta theae, Phyllosticta theicola, Phymatotrichopsisomnivora, Physalospora abdita, Physalospora disrupta, Physalosporaperseae, Physarum cinereum, Physoderma alfalfae, Physoderma leproides,Physoderma trifolii, Physopella ampelopsidis, Phytophthora, Phytophthoraalni, Phytophthora boehmeriae, Phytophthora cactorum, Phytophthoracajani, Phytophthora cambivora, Phytophthora capsici, Phytophthoracinnamomi, Phytophthora citricola, Phytophthora citrophthora,Phytophthora cryptogea, Phytophthora drechsleri, Phytophthoraerythroseptica, Phytophthora fragariae, Phytophthora fragariae var.rubi, Phytophthora gallica, Phytophthora hibernalis, Phytophthorainfestans, Phytophthora inflata, Phytophthora iranica, Phytophthorakatsurae, Phytophthora kernoviae, Phytophthora lateralis, Phytophthoramedicaginis, Phytophthora megakarya, Phytophthora megasperma,Phytophthora nicotianae, Phytophthora palmivora, Phytophthora phaseoli,Phytophthora plurivora, Phytophthora ramorum, Phytophthora sojae,Phytophthora syringae, Phytophthora tentaculata, Phytoplasma, Pichiamembranifaciens, Pichia subpelliculosa, Pileolaria terebinthi,Pilidiella quercicola, Plasmodiophora brassicae, Plasmopara, Plasmoparahalstedii, Plasmopara helianthi f. helianthi, Plasmoparalactucae-radicis, Plasmopara nivea, Plasmopara obducens, Plasmoparapenniseti, Plasmopara pygmaea, Plasmopara viticola, Platychora ulmi,Plenodomus destruens, Plenodomus meliloti, Pleochaeta, Pleosphaerulinasojicola, Pleospora alfalfae, Pleospora betae, Pleospora herbarum,Pleospora lycopersici, Pleospora tarda, Pleospora theae, Pleurotusdryinus, Podosphaera, Podosphaera clandestina var. clandestine,Podosphaera fusca, Podosphaera leucotricha, Podosphaera macularis,Podosphaera pannosa, Podosphaera tridactyla, Podosphaera tridactyla var.tridactyla, Podosphaera xanthii, Polymyxa graminis, Polyscytalumpustulans, Polystigma fulvum, Poria hypobrunnea, Postia tephroleuca,Potato cyst nematode, Pratylenchus alleni, Pratylenchus brachyurus,Pratylenchus coffeae, Pratylenchus crenatus, Pratylenchus dulscus,Pratylenchus fallax, Pratylenchus flakkensis, Pratylenchus goodeyi,Pratylenchus hexincisus, Pratylenchus loosi, Pratylenchus minutus,Pratylenchus mulchandi, Pratylenchus musicola, Pratylenchus neglectus,Pratylenchus penetrans, Pratylenchus pratensis, Pratylenchus reniformia,Pratylenchus scribneri, Pratylenchus thornei, Pratylenchus vulnus,Pratylenchus zeae, Pseudocercospora, Pseudocercospora arecacearum,Pseudocercospora cannabina, Pseudocercospora fuligena, Pseudocercosporagunnerae, Pseudocercospora kaki, Pseudocercospora mali, Pseudocercosporapandoreae, Pseudocercospora puderi, Pseudocercospora purpurea,Pseudocercospora rhapisicola, Pseudocercospora subsessilis,Pseudocercospora theae, Pseudocercospora vitis, Pseudocercosporellacapsellae, Pseudocochliobolus eragrostidis, Pseudoepicoccum cocos,Pseudomonas amygdali, Pseudomonas asplenii, Pseudomonas avellanae,Pseudomonas caricapapayae, Pseudomonas cichorii, Pseudomonascoronafaciens, Pseudomonas corrugate, Pseudomonas ficuserectae,Pseudomonas flavescens, Pseudomonas fuscovaginae, Pseudomonas helianthi,Pseudomonas marginalis, Pseudomonas meliae, Pseudomonas oryzihabitans,Pseudomonas palleroniana, Pseudomonas papaveris, Pseudomonas salomonii,Pseudomonas savastanoi, Pseudomonas syringae, Pseudomonas tomato,Pseudomonas tremae, Pseudomonas turbinellae, Pseudomonas viridiflava,Pseudoperonospora cannabina, Pseudoperonospora cubensis,Pseudoperonospora humuli, Pseudopezicula tetraspora, Pseudopeziculatracheiphila, Pseudopeziza jonesii, Pseudopeziza medicaginis,Pseudopeziza trifolii, Pseudoseptoria donacis, Puccinia, Pucciniaangustata, Puccinia arachidis, Puccinia aristidae, Puccinia asparagi,Puccinia cacabata, Puccinia campanulae, Puccinia carthami, Pucciniacoronate, Puccinia coronata var. hordei, Puccinia dioicae, Pucciniaerianthi, Puccinia extensicola var. hieraciata, Puccinia helianthi,Puccinia hordei, Puccinia jaceae var. solstitialis, Puccinia kuehnii,Puccinia mariae-wilsoniae, Puccinia melanocephala, Puccinia menthae,Puccinia pelargonii-zonalis, Puccinia pittieriana, Puccinia poarum,Puccinia psidii, Puccinia purpurea, Puccinia recondita, Pucciniaschedonnardii, Puccinia sessilis, Puccinia striiformis f. sp. hordei,Puccinia striiformis var. striiformis, Puccinia subnitens, Pucciniasubstriata var. indica, Puccinia verruca, Puccinia xanthii,Pucciniaceae, Pucciniastrum, Pucciniastrum americanum, Pucciniastrumarcticum, Pucciniastrum coryli, Pucciniastrum epilobii, Pucciniastrumhydrangeae, Punctodera chalcoensis, Pycnoporus cinnabarinus, Pycnoporussanguineus, Pycnostysanus azaleae, Pyrenochaeta lycopersici,Pyrenochaeta terrestris, Pyrenopeziza brassicae, Pyrenophora,Pyrenophora avenae, Pyrenophora chaetomioides, Pyrenophora graminea,Pyrenophora seminiperda, Pyrenophora teres, Pyrenophora teres f.maculata, Pyrenophora teres f. teres, Pyrenophora tritici-repentis,Pythiaceae, Pythiales, Pythium, Pythium acanthicum, Pythiumaphanidermatum, Pythium aristosporum, Pythium arrhenomanes, Pythiumbuismaniae, Pythium debaryanum, Pythium deliense, Pythium dissotocum,Pythium graminicola, Pythium heterothallicum, Pythium hypogynum, Pythiumirregulare, Pythium iwayamae, Pythium mastophorum, Pythium middletonii,Pythium myriotylum, Pythium okanoganense, Pythium paddicum, Pythiumparoecandrum, Pythium perniciosum, Pythium rostratum, Pythiumscleroteichum, Pythium spinosum, Pythium splendens, Pythium sulcatum,Pythium sylvaticum, Pythium tardicrescens, Pythium tracheiphilum,Pythium ultimum, Pythium ultimum var. ultimum, Pythium vexans, Pythiumviolae, Pythium volutum, Quinisulcius acutus, Quinisulcius capitatus,Radopholous similis, Radopholus similis, Ralstonia solanacearum,Ramichloridium musae, Ramularia, Ramularia beticola, Ramularia brunnea,Ramularia coryli, Ramularia cyclaminicola, Ramularia macrospora,Ramularia menthicola, Ramularia necator, Ramularia primulae, Ramulariaspinaciae, Ramularia subtilis, Ramularia tenella, Ramulispora sorghi,Ramulispora sorghicola, Resinicium bicolor, Rhabdocline pseudotsugae,Rhabdocline weirii Rhabdoviridae, Rhinocladium corticola, Rhizoctonia,Rhizoctonia leguminicola, Rhizoctonia rubi, Rhizoctonia solani,Rhizomorpha subcorticalis, Rhizophydium graminis, Rhizopus arrhizus,Rhizopus circinans, Rhizopus microsporus var. microspores, Rhizopusoryzae, Rhodococcus fascians, Rhynchosporium, Rhynchosporium secalis,Rhytidhysteron rufulum, Rhytisma acerinum, Rhytisma vitis, Rigidoporuslineatus, Rigidoporus microporus, Rigidoporus ulmarius, Rigidoporusvinctus, Rosellinia arcuata, Rosellinia bunodes, Rosellinia necatrix,Rosellinia pepo, Rosellinia subiculata, Rotylenchulus, Rotylenchulusparvus, Rotylenchulus reniformis, Rotylenchus brachyurus, Rotylenchusrobustus, Saccharicola taiwanensis, Saccharomyces florentinus,Saccharomyces kluyveri, Sarocladium oryzae, Sawadaea, Sawadaea tulasnei,Schiffnerula cannabis, Schizoparme straminea, Schizophyllum commune,Schizopora flavipora, Schizothyrium pomi, Scleroderris canker,Sclerophthora macrospora, Sclerophthora rayssiae, Sclerosporagraminicola, Sclerospora mischanthi, Sclerotinia borealis, Sclerotiniaminor, Sclerotinia ricini, Sclerotinia sclerotiorum, Sclerotiniaspermophila, Sclerotinia trifoliorum, Sclerotium, Sclerotium cinnamomi,Sclerotium delphinii, Scutellonema brachyurum, Scutellonema cavenessi,Scytinostroma galactinum, Seimatosporium mariae, Seimatosporiumrhododendri, Selenophoma linicola, Septobasidium, Septobasidiumbogoriense, Septobasidium pilosum, Septobasidium pseudopedicellatum,Septobasidium theae, Septocyta ruborum, Septogloeum potentillae,Septoria, Septoria aciculosa, Septoria ampelina, Septoria azalea,Septoria bataticola, Septoria campanulae, Septoria cannabis, Septoriacaryae, Septoria citri, Septoria cucurbitacearum, Septoria darrowii,Septoria dianthi, Septoria eumusae, Septoria fragariae, Septoriafragariaecola, Septoria glycines, Septoria helianthi, Septoria humuli,Septoria hydrangeae, Septoria lactucae, Septoria liquidambaris, Septorialycopersici, Septoria lycopersici var. malagutii, Septoria menthae,Septoria ostryae, Septoria passerinii, Septoria pisi, Septoriapistaciae, Septoria platanifolia, Septoria rhododendri, Septoriasecalis, Septoria selenophomoides, Setosphaeria rostrata, Setosphaeriaturcica, Sirosporium diffusum, Sparassis, Sphaceloma, Sphacelomaarachidis, Sphaceloma coryli, Sphaceloma menthae, Sphaceloma perseae,Sphaceloma poinsettiae, Sphaceloma pyrinum, Sphaceloma randii,Sphaceloma sacchari, Sphaceloma theae, Sphacelotheca reiliana,Sphaerella platanifolia, Sphaeropsis tumefaciens, Sphaerotheca,Sphaerotheca castagnei, Sphaerotheca fuliginea, Sphaerulina oryzina,Sphaerulina rehmiana, Sphaerulina rubi, Sphenospora kevorkianii,Spiniger meineckellus, Spiroplasma, Spongipellis unicolor, Sporisoriumcruentum, Sporisorium ehrenbergi, Sporisorium scitamineum, Sporisoriumsorghi, Sporonema phacidioides, Stagonospora avenae f.sp. triticae,Stagonospora meliloti, Stagonospora recedens, Stagonospora sacchari,Stagonospora tainanensis, Steccherinum ochraceum, Stegocintractia junci,Stegophora ulmea, Stemphylium alfalfa, Stemphylium bolickii, Stemphyliumcannabinum, Stemphylium globuliferum, Stemphylium lycopersici,Stemphylium sarciniforme, Stemphylium solani, Stemphylium vesicarium,Stenella anthuriicola, Stereum, Stereum hirsutum, Stereum rameale,Stereum sanguinolentum, Stigmatomycosis, Stigmella platani-racemosae,Stigmina carpophila, Stigmina liquidambaris, Stigmina palmivora,Stigmina platani, Stigmina platani-racemosae, Subanguina radicicola,Subanguina wevelli, Sydowia polyspora, Sydowiella depressula,Sydowiellaceae, Synchytrium endobioticum, Synchytrium fragariae,Synchytrium liquidambaris, Taiwanofungus camphoratus, Tapesia acuformis,Tapesia yallundae, Taphrina aurea, Taphrina bullata, Taphrinacaerulescens, Taphrina coryli, Taphrina deformans, Taphrina entomospora,Taphrina johansonii, Taphrina potentillae, Taphrina ulmi, Taphrinawiesneri, Thanatephorus cucumeris, Thielaviopsis, Thielaviopsisbasicola, Thyrostroma compactum, Tilletia barclayana, Tilletia caries,Tilletia controversa, Tilletia laevis, Tilletia tritici, Tilletiawalkeri, Tilletiariaceae, Tobacco necrosis virus, Togniniaceae,Trachysphaera fructigena, Trametes gibbosa, Trametes hirsute, Trametesnivosa, Trametes pubescens, Tranzschelia discolor f.sp. persica,Tranzschelia pruni-spinosae var. discolor, Trichaptum biforme,Trichoderma harzianum, Trichoderma koningii, Trichoderma viride,Trichothecium roseum, Tripospermum acerinum, Truncatella, Truncatellalaurocerasi, Tubercularia lateritic, Tubercularia ulmea, Tubeufiapezizula, Tunstallia aculeata, Tylenchorhynchus, Tylenchorhynchusbrevilineatus, Tylenchorhynchus claytoni, Tylenchorhynchus dubius,Tylenchorhynchus maximus, Tylenchorhynchus nudus, Tylenchorhynchusphaseoli, Tylenchorhynchus vulgaris, Tylenchorhynchus zeae, Tylenchulussemipenetrans, Typhula idahoensis, Typhula incarnate, Typhulaishikariensis, Typhula ishikariensis var. canadensis, Typhulavariabilis, Typhulochaeta, Tyromyces calkinsii, Tyromyces chioneus,Tyromyces galactinus, Ulocladium atrum, Ulocladium consortiale,Uncinula, Uncinula macrospora, Uncinula necator, Uredo behnickiana,Uredo kriegeriana, Uredo musae, Uredo nigropuncta, Uredo rangelii,Urocystis, Urocystis agropyri, Urocystis brassicae, Urocystis occulta,Uromyces, Uromyces apiosporus, Uromyces beticola, Uromycesciceris-arietini, Uromyces dianthi, Uromyces euphorbiae, Uromycesgraminis, Uromyces inconspicuus, Uromyces lineolatus subsp. nearcticus,Uromyces medicaginis, Uromyces musae, Uromyces oblongus, Uromycespisi-sativi, Uromyces proeminens var. poinsettiae, Uromycestrifolii-repentis var. fallen, Uromyces viciae-fabae var. viciae-fabae,Urophlyctis leproides, Urophlyctis trifolii, Urophora cardui,Ustilaginales, Ustilaginoidea virens, Ustilaginomycetes, Ustilago,Ustilago avenae, Ustilago hordei, Ustilago maydis, Ustilago nigra,Ustilago nuda, Ustilago scitaminea, Ustilago tritici, Valsa abietis,Valsa ambiens, Valsa auerswaldii, Valsa ceratosperma, Valsa kunzei,Valsa nivea, Valsa sordida, Valsaria insitiva, Venturia carpophila,Venturia inaequalis, Venturia pirina, Venturia pyrina, Veronaea musae,Verticillium, Verticillium albo-atrum, Verticillium albo-atrum var.menthae, Verticillium dahliae, Verticillium longisporum, Verticilliumtheobromae, Villosiclava virens, Virescence, Waitea circinate,Wuestneiopsis Georgiana, Xanthomonas ampelina, Xanthomonas axonopodis,Xanthomonas campestris, Xanthomonas campestris pv. campestris,Xanthomonas oryzae, Xeromphalina fraxinophila, Xiphinema americanum,Xiphinema bakeri, Xiphinema brevicolle, Xiphinema diversicaudatum,Xiphinema insigne, Xiphinema rivesi, Xiphinema vuittenezi, Xylaria mali,Xylaria polymorphs, Xylella fastidiosa, Xylophilus, Xylophilusampelinus, Zopfia rhizophila, Zygosaccharomyces bailii, andZygosaccharomyces fiorentinus.

Root Binding Proteins and Peptides

The invention also relates to fusion proteins comprising a targetingsequence and at least one root binding protein or peptide. The rootbinding protein or peptide can be any protein or peptide that is capableof specifically or non-specifically binding to a plant root.

Suitable root binding proteins and peptides include adhesins (e.g.,rhicadhesin), flagellins, omptins, lectins, pilus proteins, curlusproteins, intimins, invasins, agglutinins, and afimbrial proteins.

Recombinant Bacillus cereus Family Members that Express the FusionProteins

The present invention also relates to a recombinant Bacillus cereusfamily member that expresses a fusion protein. The fusion protein can beany of the fusion proteins discussed above.

The recombinant Bacillus cereus family member can coexpress two or moreof any of the fusion proteins discussed above. For example, therecombinant Bacillus cereus family member can coexpress at least onefusion protein that comprises a root binding protein or peptide,together with at least one fusion protein comprising a plant growthstimulating protein or peptide or at least one fusion protein comprisinga protein or peptide that protects a plant from a pathogen.

The recombinant Bacillus cereus family member can comprise Bacillusanthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides,Bacillus pseudomycoides, Bacillus weihenstephensis, or a combinationthereof.

To generate a recombinant Bacillus cereus family member expressing afusion protein, any Bacillus cereus family member can be conjugated,transduced, or transformed with a vector encoding the fusion proteinusing standard methods known in the art (e.g., by electroporation). Thebacteria can then be screened to identify transformants by any methodknown in the art. For example, where the vector includes an antibioticresistance gene, the bacteria can be screened for antibiotic resistance.The recombinant Bacillus cereus family member can then exposed toconditions which will induce sporulation. Suitable conditions forinducing sporulation are known in the art. For example, the recombinantBacillus cereus family member can be plated onto agar plates, andincubated at a temperature of about 30° C. for several days (e.g., 3days).

Inactivated strains, non-toxic strains, or genetically manipulatedstrains of any of the above species can also suitably be used. Forexample, a Bacillus thuringiensis that lacks the Cry toxin can be used.Alternatively or in addition, once the recombinant B. cereus familyspores expressing the fusion protein have been generated, they can beinactivated to prevent further germination once in use. Any method forinactivating bacterial spores that is known in the art can be used.Suitable methods include, without limitation, UV exposure, heattreatment, and irradiation. Alternatively, spores derived fromnontoxigenic strains, or genetically or physically inactivated strains,can be used.

Formulations

The present invention also relates to formulations comprising any of therecombinant Bacillus cereus family members discussed in the precedingsection and an agriculturally acceptable carrier.

The agriculturally acceptable carrier can be any carrier suitable foragricultural use. For example, suitable agriculturally acceptablecarriers include, but are not limited to dispersants, surfactants,additives, water, thickeners, anti-caking agents, residue breakdown,composting formulations, granular applications, diatomaceous earth,oils, coloring agents, stabilizers, preservatives, polymers, coatings,and combinations thereof.

The additive can comprise an oil, a gum, a resin, a clay, apolyoxyethylene glycol, a terpene, a viscid organic, a fatty acid ester,a sulfated alcohol, an alkyl sulfonate, a petroleum sulfonate, analcohol sulfate, a sodium alkyl butane diamate, a polyester of sodiumthiobutane dioate, a benzene acetonitrile derivative, a proteinaceousmaterial (e.g., a milk product, wheat flour, soybean meal, blood,albumin, gelatin, or a combination thereof), or a combination thereof.

The thickener can comprise a long chain alkylsulfonate of polyethyleneglycol, a polyoxyethylene oleate, or a combination thereof.

The surfactant can comprise a heavy petroleum oil, a heavy petroleumdistillate, a polyol fatty acid ester, a polyethoxylated fatty acidester, an aryl alkyl polyoxyethylene glycol, an alkyl amine acetate, analkyl aryl sulfonate, a polyhydric alcohol, an alkyl phosphate, or acombination thereof.

The anti-caking agent comprises a sodium salt, a calcium carbonate, asodium sulfite, a sodium sulfate, diatomaceous earth, or a combinationthereof. For example, the sodium salt can comprise a sodium salt ofmonomethyl naphthalene sulfonate, a sodium salt of dimethyl naphthalenesulfonate, or a combination thereof.

Suitable agriculturally acceptable carriers include vermiculite,charcoal, sugar factory carbonation press mud, rice husk, carboxymethylcellulose, peat, perlite, fine sand, calcium carbonate, flour, alum, astarch, talc, polyvinyl pyrrolidone, or a combination thereof.

The formulation can comprise a seed coating formulation, a liquidformulation for application to plants or to a plant growth medium, or asolid formulation for application to plants or to a plant growth medium.

For example, the seed coating formulation can comprise an aqueous oroil-based solution for application to seeds. Alternatively, the seedcoating formulation can comprise a powder or granular formulation forapplication to seeds.

The liquid formulation for application to plants or to a plant growthmedium can comprise a concentrated formulation or a working formformulation.

The solid formulation for application to plants or to a plant growthmedium can comprises a granular formulation or a powder agent.

Any of the above formulations can also comprise an agrochemical, forexample, a fertilizer, a micronutrient fertilizer material, aninsecticide, a herbicide, a plant growth amendment, a fungicide, aninsecticide, a molluscicide, an algicide, a bacterial inoculant, afungal inoculant, or a combination thereof.

The fertilizer can comprise a liquid fertilizer.

The fertilizer can comprise ammonium sulfate, ammonium nitrate, ammoniumsulfate nitrate, ammonium chloride, ammonium bisulfate, ammoniumpolysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia,ammonium polyphosphate, aluminum sulfate, calcium nitrate, calciumammonium nitrate, calcium sulfate, calcined magnesite, calciticlimestone, calcium oxide, calcium nitrate, dolomitic limestone, hydratedlime, calcium carbonate, diammonium phosphate, monoammonium phosphate,magnesium nitrate, magnesium sulfate, potassium nitrate, potassiumchloride, potassium magnesium sulfate, potassium sulfate, sodiumnitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, ureaammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylidenediurea, K₂SO₄-2MgSO₄, kainite, sylvinite, kieserite, Epsom salts,elemental sulfur, marl, ground oyster shells, fish meal, oil cakes, fishmanure, blood meal, rock phosphate, super phosphates, slag, bone meal,wood ash, manure, bat guano, peat moss, compost, green sand, cottonseedmeal, feather meal, crab meal, fish emulsion, or a combination thereof.

The micronutrient fertilizer material can comprise boric acid, a borate,a boron frit, copper sulfate, a copper frit, a copper chelate, a sodiumtetraborate decahydrate, an iron sulfate, an iron oxide, iron ammoniumsulfate, an iron frit, an iron chelate, a manganese sulfate, a manganeseoxide, a manganese chelate, a manganese chloride, a manganese frit, asodium molybdate, molybdic acid, a zinc sulfate, a zinc oxide, a zinccarbonate, a zinc frit, zinc phosphate, a zinc chelate, or a combinationthereof.

The insecticide can comprise an organophosphate, a carbamate, apyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, afluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester,a biologically-based insecticide, or a combination thereof.

The herbicide can comprise a chlorophenoxy compound, a nitrophenoliccompound, a nitrocresolic compound, a dipyridyl compound, an acetamide,an aliphatic acid, an anilide, a benzamide, a benzoic acid, a benzoicacid derivative, anisic acid, an anisic acid derivative, a benzonitrile,benzothiadiazinone dioxide, a thiocarbamate, a carbamate, a carbanilate,chloropyridinyl, a cyclohexenone derivative, a dinitroaminobenzenederivative, a fluorodinitrotoluidine compound, isoxazolidinone,nicotinic acid, isopropylamine, an isopropylamine derivatives,oxadiazolinone, a phosphate, a phthalate, a picolinic acid compound, atriazine, a triazole, a uracil, a urea derivative, endothall, sodiumchlorate, or a combination thereof.

The fungicide can comprise a substituted benzene, a thiocarbamate, anethylene bis dithiocarbamate, a thiophthalidamide, a copper compound, anorganomercury compound, an organotin compound, a cadmium compound,anilazine, benomyl, cyclohexamide, dodine, etridiazole, iprodione,metlaxyl, thiamimefon, triforine, or a combination thereof.

The fungal inoculant can comprise a fungal inoculant of the familyGlomeraceae, a fungal inoculant of the family Claroidoglomeraceae, afungal inoculant of the family Gigasporaceae, a fungal inoculant of thefamily Acaulosporaceae, a fungal inoculant of the familySacculosporaceae, a fungal inoculant of the family Entrophosphoraceae, afungal inoculant of the family Pacidsporaceae, a fungal inoculant of thefamily Diversisporaceae, a fungal inoculant of the familyParaglomeraceae, a fungal inoculant of the family Archaeosporaceae, afungal inoculant of the family Geosiphonaceae, a fungal inoculant of thefamily Ambisporaceae, a fungal inoculant of the familyScutellosporaceae, a fungal inoculant of the family Dentiscultataceae, afungal inoculant of the family Racocetraceae, a fungal inoculant of thephylum Basidiomycota, a fungal inoculant of the phylum Ascomycota, afungal inoculant of the phylum Zygomycota, or a combination thereof.

The bacterial inoculant can comprise a bacterial inoculant of the genusRhizobium, a bacterial inoculant of the genus Bradyrhizobium, abacterial inoculant of the genus Mesorhizobium, a bacterial inoculant ofthe genus Azorhizobium, a bacterial inoculant of the genusAllorhizobium, a bacterial inoculant of the genus Sinorhizobium, abacterial inoculant of the genus Kluyvera, a bacterial inoculant of thegenus Azotobacter, a bacterial inoculant of the genus Pseudomonas, abacterial inoculant of the genus Azospirillium, a bacterial inoculant ofthe genus Bacillus, a bacterial inoculant of the genus Streptomyces, abacterial inoculant of the genus Paenibacillus, a bacterial inoculant ofthe genus Paracoccus, a bacterial inoculant of the genus Enterobacter, abacterial inoculant of the genus Alcaligenes, a bacterial inoculant ofthe genus Mycobacterium, a bacterial inoculant of the genus Trichoderma,a bacterial inoculant of the genus Gliocladium, a bacterial inoculant ofthe genus Glomus, a bacterial inoculant of the genus Klebsiella, or acombination thereof.

Methods for Promoting Plant Growth

The present invention also relates to methods for stimulating plantgrowth. The method for stimulating plant growth comprises introducinginto a plant growth medium any of the recombinant Bacillus cereus familymembers discussed above or any of the formulations discussed above.Alternatively, any of the recombinant Bacillus cereus family membersdiscussed above or any of the formulations discussed above can beapplied to the foliage of a plant, to a plant seed, or to an areasurrounding a plant. In such methods, the plant growth stimulatingprotein or peptide is physically attached to the exosporium of therecombinant Bacillus family member.

Alternatively, the method for stimulating plant growth comprisesintroducing a recombinant Bacillus cereus family member expressing afusion protein into a plant growth medium or applying a recombinantBacillus cereus family member expressing a fusion protein to foliage ofa plant, a plant seed, or an area surrounding a plant. The fusionprotein comprises at least one plant growth stimulating protein orpeptide and a targeting sequence. The targeting sequence can be any ofthe targeting sequences discussed herein.

The plant growth stimulating protein can comprise an enzyme. Forexample, the enzyme can comprise an enzyme that degrades or modifies abacterial, fungal, or plant nutrient source. Such enzymes includecellulases, lipases, lignin oxidases, proteases, glycoside hydrolases,phosphatases, nitrogenases, and nucleases.

Suitable cellulases include endocellulases (e.g., a Bacillus subtilisendoglucanase, a Bacillus thuringiensis endoglucanase, a Bacillus cereusendoglucanase, or a Bacillus clausii endoglucanase), exocellulases(e.g., a Trichoderma reesei exocellulase), and β-glucosidases (e.g., aBacillus subtilis β-glucosidase, a Bacillus thuringiensis β-glucosidase,a Bacillus cereus β-glucosidase, or a Bacillus clausii β-glucosidase).

The lipase can comprise a Bacillus subtilis lipase, a Bacillusthuringiensis lipase, a Bacillus cereus lipase, or a Bacillus clausiilipase.

Suitable lignin oxidases comprise lignin peroxidases, laccases, glyoxaloxidases, liginases, and manganese peroxidases.

The protease can comprise a subtilisin, an acid protease, an alkalineprotease, a proteinase, a peptidase, an endopeptidase, an exopeptidase,a thermolysin, a papain, a pepsin, a trypsin, a pronase, a carboxylase,a serine protease, a glutamic protease, an aspartate protease, acysteine protease, a threonine protease, or a metalloprotease.

The phosphatase can comprise a phosphoric monoester hydrolase, aphosphomonoesterase, a phosphoric diester hydrolase, aphosphodiesterase, a triphosphoric monoester hydrolase, a phosphorylanhydride hydrolase, a pyrophosphatase, a phytase, a trimetaphosphatase,or a triphosphatase.

The nitrogenase can comprise a Nif family nitrogenase.

In any of the above methods for stimulating plant growth, plants grownin the plant growth medium comprising the recombinant Bacillus cereusfamily member exhibit increased growth as compared to the growth ofplants in the identical plant growth medium that does not contain therecombinant Bacillus cereus family member.

Methods for Protecting a Plant from a Pathogen

The present invention further relates to methods for protecting a plantfrom a pathogen. Such methods comprise introducing any of therecombinant Bacillus cereus family members discussed above or any of theformulations discussed above into a plant growth medium. Alternatively,such methods comprise applying any of the recombinant Bacillus cereusfamily members discussed above or any of the formulations discussedabove to foliage of a plant, to a plant seed, or to an area surroundinga plant. In these methods, the protein or peptide that protects a plantfrom a pathogen is physically attached to the exosporium of therecombinant Bacillus cereus family member.

Plants grown in the plant growth medium comprising the recombinantBacillus cereus family member are less susceptible to infection with thepathogen as compared to plants grown in the identical plant growthmedium that does not contain the recombinant Bacillus cereus familymember.

Methods for Immobilizing Bacillus Spores on a Root System

The present invention is also directed to methods for immobilizing arecombinant Bacillus cereus family member spore on a root system of aplant. These methods comprise introducing any of the recombinantBacillus cereus family members discussed above or any of theformulations discussed above into a plant growth medium. Alternatively,such methods comprise applying any of the recombinant Bacillus cereusfamily members discussed above or any of the formulations discussedabove to foliage of a plant, to a plant seed, or to an area surroundinga plant. The root binding protein or peptide is physically attached tothe exosporium of the recombinant Bacillus family member.

These methods allow the Bacillus cereus family member spore to bind to aroot of a plant, such that the spore is maintained at the plant's rootstructure instead of dissipating into the plant growth medium.

In any of the methods for immobilizing a recombinant Bacillus cereusfamily member spore on a root system of a plant, the root bindingprotein or peptide can selectively target and maintain the Bacilluscereus family member at plant roots and substructures of plant roots.

Plant Growth Medium

In any of the above methods, the plant growth medium is material that iscapable of supporting the growth of a plant. The plant growth medium cancomprise soil, water, an aqueous solution, sand, gravel, apolysaccharide, mulch, compost, peat moss, straw, logs, clay, soybeanmeal, yeast extract, or a combination thereof. For example, the plantgrowth medium comprises soil, compost, peat moss, or a combinationthereof.

The plant growth medium can optionally be supplemented with a substratefor an enzyme. For example, the substrate can comprise tryptophan, anadenosine monophosphate, an adenosine diphosphate, an adenosinetriphosphate (e.g., adenosine-3-triphosphate), indole, atrimetaphosphate, ferrodoxin, acetoin, diacetyl, pyruvate, acetolactate,or a combination thereof.

Application Methods

In any of the above methods, the recombinant Bacillus cereus familymember or formulation can be introduced into the plant growth medium orapplied to foliage of a plant, to a plant seed, or to an areasurrounding a plant.

For example, the method can comprise coating seeds with the recombinantBacillus cereus family member or a formulation containing therecombinant Bacillus cereus family member prior to planting.

Alternatively, the method can comprise applying the recombinant Bacilluscereus family member or formulation to plant foliage.

The method can comprise introducing the recombinant Bacillus cereusfamily member into the plant growth medium by applying a liquid or solidformulation containing the recombinant Bacillus cereus family member tothe medium (e.g., soil, compost, peat moss, or a combination thereof).

The formulation can be applied to the plant growth medium prior to,concurrently with, or after planting of seeds, seedlings, cuttings,bulbs, or plants in the plant growth medium.

Co-Application of Agrochemicals

Any of the above methods can further comprise introducing at least oneagrochemical into the plant growth medium or applying at least oneagrochemical to plants or seeds. The agrochemical can be any of thoselisted above for inclusion in the formulations, or any combinationthereof.

Plants

The above methods can be practiced with a variety of plants. Forexample, the plant can be a dicotyledon, a monocotyledon, or agymnosperm.

For example, where the plant is a dicotyledon, the dicotyledon can beselected from the group consisting of bean, pea, tomato, pepper, squash,alfalfa, almond, aniseseed, apple, apricot, arracha, artichoke, avocado,bambara groundnut, beet, bergamot, black pepper, black wattle,blackberry, blueberry, bitter orange, bok-choi, Brazil nut, breadfruit,broccoli, broad bean, Brussels sprouts, buckwheat, cabbage, camelina,Chinese cabbage, cacao, cantaloupe, caraway seeds, cardoon, carob,carrot, cashew nuts, cassava, castor bean, cauliflower, celeriac,celery, cherry, chestnut, chickpea, chicory, chili pepper,chrysanthemum, cinnamon, citron, clementine, clove, clover, coffee, colanut, colza, corn, cotton, cottonseed, cowpea, crambe, cranberry, cress,cucumber, currant, custard apple, drumstick tree, earth pea, eggplant,endive, fennel, fenugreek, fig, filbert, flax, geranium, gooseberry,gourd, grape, grapefruit, guava, hemp, hempseed, henna, hop, horse bean,horseradish, indigo, jasmine, Jerusalem artichoke, jute, kale, kapok,kenaf, kohlrabi, kumquat, lavender, lemon, lentil, lespedeza, lettuce,lime, liquorice, litchi, loquat, lupine, macadamia nut, mace, mandarin,mangel, mango, medlar, melon, mint, mulberry, mustard, nectarine, nigerseed, nutmeg, okra, olive, opium, orange, papaya, parsnip, pea, peach,peanut, pear, pecan nut, persimmon, pigeon pea, pistachio nut, plantain,plum, pomegranate, pomelo, poppy seed, potato, sweet potato, prune,pumpkin, quebracho, quince, trees of the genus Cinchona, quinoa, radish,ramie, rapeseed, raspberry, rhea, rhubarb, rose, rubber, rutabaga,safflower, sainfoin, salsify, sapodilla, Satsuma, scorzonera, sesame,shea tree, soybean, spinach, squash, strawberry, sugar beet, sugarcane,sunflower, swede, sweet pepper, tangerine, tea, teff, tobacco, tomato,trefoil, tung tree, turnip, urena, vetch, walnut, watermelon, yerbamate, wintercress, shepherd's purse, garden cress, peppercress,watercress, pennycress, star anise, laurel, bay laurel, cassia, jamun,dill, tamarind, peppermint, oregano, rosemary, sage, soursop, pennywort,calophyllum, balsam pear, kukui nut, Tahitian chestnut, basil,huckleberry, hibiscus, passionfruit, star apple, sassafras, cactus, St.John's wort, loosestrife, hawthorn, cilantro, curry plant, kiwi, thyme,zucchini, ulluco, jicama, waterleaf, spiny monkey orange, yellow mombin,starfruit, amaranth, wasabi, Japanese pepper, yellow plum, mashua,Chinese toon, New Zealand spinach, bower spinach, ugu, tansy, chickweed,jocote, Malay apple, paracress, sowthistle, Chinese potato, horseparsley, hedge mustard, campion, agate, cassod tree, thistle, burnet,star gooseberry, saltwort, glasswort, sorrel, silver lace fern, collardgreens, primrose, cowslip, purslane, knotgrass, terebinth, tree lettuce,wild betel, West African pepper, yerba santa, tarragon, parsley,chervil, land cress, burnet saxifrage, honeyherb, butterbur, shiso,water pepper, perilla, bitter bean, oca, kampong, Chinese celery, lemonbasil, Thai basil, water mimosa, cicely, cabbage-tree, moring a, mauka,ostrich fern, rice paddy herb, yellow sawah lettuce, lovage, peppergrass, maca, bottle gourd, hyacinth bean, water spinach, catsear,fishwort, Okinawan spinach, lotus sweetjuice, gallant soldier, culantro,arugula, cardoon, caigua, mitsuba, chipilin, samphire, mampat, ebolo,ivy gourd, cabbage thistle, sea kale, chaya, huauzontle, Ethiopianmustard, magenta spreen, good king henry, epazole, lamb's quarters,centella plumed cockscomb, caper, rapini, napa cabbage, mizuna, Chinesesavoy, kai-lan, mustard greens, Malabar spinach, chard, marshmallow,climbing wattle, China jute, paprika, annatto seed, spearmint, savory,marjoram, cumin, chamomile, lemon balm, allspice, bilberry, cherimoya,cloudberry, damson, pitaya, durian, elderberry, feijoa, jackfruit,jambul, jujube, physalis, purple mangosteen, rambutan, redcurrant,blackcurrant, salal berry, satsuma, ugli fruit, azuki bean, black bean,black-eyed pea, borlotti bean, common bean, green bean, kidney bean,lima bean, mung bean, navy bean, pinto bean, runner bean, mangetout,snap pea, broccoflower, calabrese, nettle, bell pepper, raddichio,daikon, white radish, skirret, tat soi, broccolini, black radish,burdock root, fava bean, broccoli raab, lablab, lupin, sterculia, velvetbeans, winged beans, yam beans, mulga, ironweed, umbrella bush,tjuntjula, wakalpulka, witchetty bush, wiry wattle, chia, beech nut,candlenut, colocynth, mamoncillo, Maya nut, mongongo, ogbono nut,paradise nut, and cempedak.

Alternatively, the dicotyledon can be from a family selected from thegroup consisting of Acanthaceae (acanthus), Aceraceae (maple),Achariaceae, Achatocarpaceae (achatocarpus), Actinidiaceae (Chinesegooseberry), Adoxaceae (moschatel), Aextoxicaceae, Aizoaceae (figmarigold), Akaniaceae, Alangiaceae, Alseuosmiaceae, Alzateaceae,Amaranthaceae (amaranth), Amborellaceae, Anacardiaceae (sumac),Ancistrocladaceae, Anisophylleaceae, Annonaceae (custard apple),Apiaceae (carrot), Apocynaceae (dogbane), Aquifoliaceae (holly),Araliaceae (ginseng), Aristolochiaceae (birthwort), Asclepiadaceae(milkweed), Asteraceae (aster), Austrobaileyaceae, Balanopaceae,Balanophoraceae (balanophora), Balsaminaceae (touch-me-not),Barbeyaceae, Barclayaceae, Basellaceae (basella), Bataceae (saltwort),Begoniaceae (begonia), Berberidaceae (barberry), Betulaceae (birch),Bignoniaceae (trumpet creeper), Bixaceae (lipstick tree), Bombacaceae(kapok tree), Boraginaceae (borage), Brassicaceae (mustard, alsoCruciferae), Bretschneideraceae, Brunelliaceae (brunellia), Bruniaceae,Brunoniaceae, Buddlejaceae (butterfly bush), Burseraceae (frankincense),Buxaceae (boxwood), Byblidaceae, Cabombaceae (water shield), Cactaceae(cactus), Caesalpiniaceae, Callitrichaceae (water starwort),Calycanthaceae (strawberry shrub), Calyceraceae (calycera),Campanulaceae (bellflower), Canellaceae (canella), Cannabaceae (hemp),Capparaceae (caper), Caprifoliaceae (honeysuckle), Cardiopteridaceae,Caricaceae (papaya), Caryocaraceae (souari), Caryophyllaceae (pink),Casuarinaceae (she-oak), Cecropiaceae (cecropia), Celastraceae(bittersweet), Cephalotaceae, Ceratophyllaceae (hornwort),Cercidiphyllaceae (katsura tree), Chenopodiaceae (goosefoot),Chloranthaceae (chloranthus), Chrysobalanaceae (cocoa plum),Circaeasteraceae, Cistaceae (rockrose), Clethraceae (clethra),Clusiaceae (mangosteen, also Guttiferae), Cneoraceae, Columelliaceae,Combretaceae (Indian almond), Compositae (aster), Connaraceae(cannarus), Convolvulaceae (morning glory), Coriariaceae, Cornaceae(dogwood), Corynocarpaceae (karaka), Crassulaceae (stonecrop),Crossosomataceae (crossosoma), Crypteroniaceae, Cucurbitaceae(cucumber), Cunoniaceae (cunonia), Cuscutaceae (dodder), Cyrillaceae(cyrilla), Daphniphyllaceae, Datiscaceae (datisca), Davidsoniaceae,Degeneriaceae, Dialypetalanthaceae, Diapensiaceae (diapensia),Dichapetalaceae, Didiereaceae, Didymelaceae, Dilleniaceae (dillenia),Dioncophyllaceae, Dipentodontaceae, Dipsacaceae (teasel),Dipterocarpaceae (meranti), Donatiaceae, Droseraceae (sundew),Duckeodendraceae, Ebenaceae (ebony), Elaeagnaceae (oleaster),Elaeocarpaceae (elaeocarpus), Elatinaceae (waterwort), Empetraceae(crowberry), Epacridaceae (epacris), Eremolepidaceae (catkin-mistletoe),Ericaceae (heath), Erythroxylaceae (coca), Eucommiaceae, Eucryphiaceae,Euphorbiaceae (spurge), Eupomatiaceae, Eupteleaceae, Fabaceae (pea orlegume), Fagaceae (beech), Flacourtiaceae (flacourtia), Fouquieriaceae(ocotillo), Frankeniaceae (frankenia), Fumariaceae (fumitory),Garryaceae (silk tassel), Geissolomataceae, Gentianaceae (gentian),Geraniaceae (geranium), Gesneriaceae (gesneriad), Globulariaceae,Gomortegaceae, Goodeniaceae (goodenia), Greyiaceae, Grossulariaceae(currant), Grubbiaceae, Gunneraceae (gunnera), Gyrostemonaceae,Haloragaceae (water milfoil), Hamamelidaceae (witch hazel),Hernandiaceae (hernandia), Himantandraceae, Hippocastanaceae (horsechestnut), Hippocrateaceae (hippocratea), Hippuridaceae (mare's tail),Hoplestigmataceae, Huaceae, Hugoniaceae, Humiriaceae, Hydnoraceae,Hydrangeaceae (hydrangea), Hydrophyllaceae (waterleaf),Hydrostachyaceae, Icacinaceae (icacina), Idiospermaceae, Illiciaceae(star anise), Ixonanthaceae, Juglandaceae (walnut), Julianiaceae,Krameriaceae (krameria), Lacistemataceae, Lamiaceae (mint, alsoLabiatae), Lardizabalaceae (lardizabala), Lauraceae (laurel),Lecythidaceae (brazil nut), Leeaceae, Leitneriaceae (corkwood),Lennoaceae (lennoa), Lentibulariaceae (bladderwort), Limnanthaceae(meadow foam), Linaceae (flax), Lissocarpaceae, Loasaceae (loasa),Loganiaceae (logania), Loranthaceae (showy mistletoe), Lythraceae(loosestrife), Magnoliaceae (magnolia), Malesherbiaceae, Malpighiaceae(barbados cherry), Malvaceae (mallow), Marcgraviaceae (shingle plant),Medusagynaceae, Medusandraceae, Melastomataceae (melastome), Meliaceae(mahogany), Melianthaceae, Mendonciaceae, Menispermaceae (moonseed),Menyanthaceae (buckbean), Mimosaceae, Misodendraceae, Mitrastemonaceae,Molluginaceae (carpetweed), Monimiaceae (monimia), Monotropaceae (Indianpipe), Moraceae (mulberry), Moringaceae (horseradish tree), Myoporaceae(myoporum), Myricaceae (bayberry), Myristicaceae (nutmeg),Myrothamnaceae, Myrsinaceae (myrsine), Myrtaceae (myrtle), Nelumbonaceae(lotus lily), Nepenthaceae (East Indian pitcherplant), Neuradaceae,Nolanaceae, Nothofagaceae, Nyctaginaceae (four-o'clock), Nymphaeaceae(water lily), Nyssaceae (sour gum), Ochnaceae (ochna), Olacaceae (olax),Oleaceae (olive), Oliniaceae, Onagraceae (evening primrose),Oncothecaceae, Opiliaceae, Orobanchaceae (broom rape), Oxalidaceae (woodsorrel), Paeoniaceae (peony), Pandaceae, Papaveraceae (poppy),Papilionaceae, Paracryphiaceae, Passifloraceae (passionflower),Pedaliaceae (sesame), Pellicieraceae, Penaeaceae, Pentaphragmataceae,Pentaphylacaceae, Peridiscaceae, Physenaceae, Phytolaccaceae (pokeweed),Piperaceae (pepper), Pittosporaceae (pittosporum), Plantaginaceae(plantain), Platanaceae (plane tree), Plumbaginaceae (leadwort),Podostemaceae (river weed), Polemoniaceae (phlox), Polygalaceae(milkwort), Polygonaceae (buckwheat), Portulacaceae (purslane),Primulaceae (primrose), Proteaceae (protea), Punicaceae (pomegranate),Pyrolaceae (shinleaf), Quiinaceae, Rafflesiaceae (rafflesia),Ranunculaceae (buttercup orranunculus), Resedaceae (mignonette),Retziaceae, Rhabdodendraceae, Rhamnaceae (buckthorn), Rhizophoraceae(red mangrove), Rhoipteleaceae, Rhynchocalycaceae, Rosaceae (rose),Rubiaceae (madder), Rutaceae (rue), Sabiaceae (sabia), Saccifoliaceae,Salicaceae (willow), Salvadoraceae, Santalaceae (sandalwood),Sapindaceae (soapberry), Sapotaceae (sapodilla), Sarcolaenaceae,Sargentodoxaceae, Sarraceniaceae (pitcher plant), Saururaceae (lizard'stail), Saxifragaceae (saxifrage), Schisandraceae (schisandra),Scrophulariaceae (figwort), Scyphostegiaceae, Scytopetalaceae,Simaroubaceae (quassia), Simmondsiaceae (jojoba), Solanaceae (potato),Sonneratiaceae (sonneratia), Sphaerosepalaceae, Sphenocleaceae(spenoclea), Stackhousiaceae (stackhousia), Stachyuraceae, Staphyleaceae(bladdernut), Sterculiaceae (cacao), Stylidiaceae, Styracaceae (storax),Surianaceae (suriana), Symplocaceae (sweetleaf), Tamaricaceae (tamarix),Tepuianthaceae, Tetracentraceae, Tetrameristaceae, Theaceae (tea),Theligonaceae, Theophrastaceae (theophrasta), Thymelaeaceae (mezereum),Ticodendraceae, Tiliaceae (linden), Tovariaceae, Trapaceae (waterchestnut), Tremandraceae, Trigoniaceae, Trimeniaceae, Trochodendraceae,Tropaeolaceae (nasturtium), Turneraceae (turnera), Ulmaceae (elm),Urticaceae (nettle), Valerianaceae (valerian), Verbenaceae (verbena),Violaceae (violet), Viscaceae (Christmas mistletoe), Vitaceae (grape),Vochysiaceae, Winteraceae (wintera), Xanthophyllaceae, andZygophyllaceae (creosote bush).

Where the plant is a monocotyledon, the monocotyledon can be selectedfrom the group consisting of corn, wheat, oat, rice, barley, millet,banana, onion, garlic, asparagus, ryegrass, millet, fonio, raishan, nipagrass, turmeric, saffron, galangal, chive, cardamom, date palm,pineapple, shallot, leek, scallion, water chestnut, ramp, Job's tears,bamboo, ragi, spotless watermeal, arrowleaf elephant ear, Tahitianspinach, abaca, areca, bajra, betel nut, broom millet, broom sorghum,citronella, coconut, cocoyam, maize, dasheen, durra, durum wheat, edo,fique, formio, ginger, orchard grass, esparto grass, Sudan grass, guineacorn, Manila hemp, henequen, hybrid maize, jowar, lemon grass, maguey,bulrush millet, finger millet, foxtail millet, Japanese millet, prosomillet, New Zealand flax, oats, oil palm, palm palmyra, sago palm,redtop, sisal, sorghum, spelt wheat, sweet corn, sweet sorghum, taro,teff, timothy grass, triticale, vanilla, wheat, and yam.

Alternatively, the monocotyledon can be from a family selected from thegroup consisting of Acoraceae (calamus), Agavaceae (century plant),Alismataceae (water plantain), Aloeaceae (aloe), Aponogetonaceae (capepondweed), Araceae (arum), Arecaceae (palm), Bromeliaceae (bromeliad),Burmanniaceae (burmannia), Butomaceae (flowering rush), Cannaceae(canna), Centrolepidaceae, Commelinaceae (spiderwort), Corsiaceae,Costaceae (costus), Cyanastraceae, Cyclanthaceae (Panama hat),Cymodoceaceae (manatee grass), Cyperaceae (sedge), Dioscoreaceae (yam),Eriocaulaceae (pipewort), Flagellariaceae, Geosiridaceae, Haemodoraceae(bloodwort), Hanguanaceae (hanguana), Heliconiaceae (heliconia),Hydatellaceae, Hydrocharitaceae (tape grass), Iridaceae (iris),Joinvilleaceae (joinvillea), Juncaceae (rush), Juncaginaceae (arrowgrass), Lemnaceae (duckweed), Liliaceae (lily), Limnocharitaceae (waterpoppy), Lowiaceae, Marantaceae (prayer plant), Mayacaceae (mayaca),Musaceae (banana), Najadaceae (water nymph), Orchidaceae (orchid),Pandanaceae (screw pine), Petrosaviaceae, Philydraceae (philydraceae),Poaceae (grass), Pontederiaceae (water hyacinth), Posidoniaceae(posidonia), Potamogetonaceae (pondweed), Rapateaceae, Restionaceae,Ruppiaceae (ditch grass), Scheuchzeriaceae (scheuchzeria), Smilacaceae(catbrier), Sparganiaceae (bur reed), Stemonaceae (stemona),Strelitziaceae, Taccaceae (tacca), Thurniaceae, Triuridaceae, Typhaceae(cattail), Velloziaceae, Xanthorrhoeaceae, Xyridaceae (yellow-eyedgrass), Zannichelliaceae (horned pondweed), Zingiberaceae (ginger), andZosteraceae (eelgrass).

Where the plant is a gymnosperm, the gymnosperm can be from a familyselected from the group consisting of Araucariaceae, Boweniaceae,Cephalotaxaceae, Cupressaceae, Cycadaceae, Ephedraceae, Ginkgoaceae,Gnetaceae, Pinaceae, Podocarpaceae, Taxaceae, Taxodiaceae,Welwitschiaceae, and Zamiaceae.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention.

Example 1 Use of a Recombinant Bacillus cereus Family Member Displayinga Lipase or an Endogluconase to Stimulate Plant Growth in Soybeans

The Bacillus subtilis lipase and endoglucanase genes were amplified viapolymerase chain reaction (PCR) using the following primers shown belowin Table 2:

TABLE 2 lipase endogluconase for- ggatccatggctgaacacaatccggatccatgaaacggtcaatc ward (SEQ ID NO: 37) (SEQ ID NO: 39) re-ggatccttaattcgtattctggc ggatccttactaatttggttc verse c tgt(SEQ ID NO: 38) (SEQ ID NO: 40)

To create fusion constructs, genes were fused to the native bclApromoter of Bacillus thuringiensis DNA encoding the first 35 amino acidsof BclA (amino acids 1-35 of SEQ ID NO:1) using the splicing byoverlapping extension (SOE) technique. Correct amplicons were clonedinto the E. coli/Bacillus shuttle vector pHP13, and correct clonesscreened by DNA sequencing. Correct clones were electroporated intoBacillus thuringiensis (Cry-, plasmid-) and screened for chloramphenicolresistance. Correct transformants were grown in Brain Heart Infusionbroth overnight at 30° C., plated onto nutrient agar plates, andincubated at 30° C. for 3 days. Spores expressing the fusion construct(BEMD spores) were collected off of the plates by washing in phosphatebuffered saline (PBS) and purified by centrifugation and additionalwashes in PBS. Non-transformed control Bacillus thuringiensis (B.t.)spores were created identically.

Soybeans (strain Jake 011-28-04) were planted 1 inch (2.54 cm) deep in10 cm deep pots filled with standard loam topsoil. Spores were dilutedto a concentration of 1×10⁴/ml in 50 ml of water and applied to eachseed at planting. Plants were grown under ideal light using T5 lamps, 54watts, and exposed to 11 hours of light a day under controlledtemperature conditions between 60-78° F. (15.5-25.5° C.). Plants werewatered to saturation every three days over a two week trial. At the endof two weeks, the height of each plant was measured and measurementswere normalized to control Bacillus thuringiensis spores. Twoindependent trials were performed.

Results are shown in Table 3, together with the standard error of themean. In both trials, soybeans grown in the presence of BEMD sporesdisplaying either lipase or endoglucanase grew significantly taller thancontrol B.t. spore treated soybeans (statistical analysis assayed via at-test).

TABLE 3 Soybeans Avg. Height, Comparison to Treatment Inches (cm)Control SEM Trial #1 Control Bt 5.525 (14.034) 100.0% .521 Lipase, BEMD7.06 (17.93) 127.8% .395 Endocellulase, BEMD 6.42 (16.31) 116.2% .411Trial #2 Control Bt 6.06 (15.39) 100.0% .749 Lipase, BEMD 7.54 (19.15)124.4% .428 Endocellulase, BEMD 6.95 (17.65) 114.7% .313

Example 2 Use of a Recombinant Bacillus cereus Family Member Displayingan Endoglucanase to Stimulate Plant Growth in Corn

BEMD spores expressing endoglucanase were created in an identicalfashion as described above in Example 1. Field corn was planted 1.5inches (3.8 cm) deep in 10 cm deep pots filled with standard loamtopsoil. Spores, control and BEMD expressing endoglucanase, were dilutedto a concentration of 1×10⁴/ml in 50 ml of water and applied to eachplant at planting. A water-only control was also included. Plants weregrown under ideal light using T5 lamps, 54 watts, and exposed to 11hours of light a day under controlled temperature conditions between60-78° F. (15.5-25.5° C.). Plants were watered to saturation every threedays over the one week trial. At the end of one week, the height of eachplant was measured, and measurements were normalized to control Bacillusthuringiensis spores.

Results are shown in Table 4, together with the standard error of themean. Corn grown in the presence of BEMD spores displaying endoglucanasegrew significantly taller than both control B.t. spore treated soybeansand water-only control plants (statistical analysis assayed via at-test).

TABLE 4 Height, inches (cm) Comparison SEM H₂O 6.08 (15.44)   100% 0.318Bt 7.45 (18.92) 122.50% 0.645 BEMD Endo 8.73 (22.71) 143.40% 0.616

Example 3 Use of a Recombinant Bacillus cereus Family Member Displayingan Endogluconase or a Protease to Stimulate Plant Growth in Wheat

BEMD spores expressing endoglucanase were created in an identicalfashion as described above in Example 1. BEMD spores expressing E. coliprotease PtrB were created using similar methods to those describedabove in Example 1 and the following primers: ggatccatgctaccaaaagcc(forward, SEQ ID NO: 41) and ggatccttagtccgcaggcgtagc (reverse, SEQ IDNO: 42).

Winter hard wheat was planted 1 inch (2.54 cm) deep in 10 cm deep potsfilled with standard loam topsoil. Spores, control and BEMD expressingendoglucanase or protease, were diluted to a concentration of 1×10⁴/mlin 50 ml of water and applied to each plant at planting. A water-onlycontrol was also included. Plants were grown under ideal light using T5lamps, 54 watts, and exposed to 11 hours of light a day under controlledtemperature conditions between 60-78° F. (15.5-25.5° C.). Plants werewatered to saturation every three days over the one week trial. At theend of one week, the height of each plant was measured, and measurementswere normalized to control water only plants.

Results are shown in Table 5, together with the standard error of themean. Wheat grown in the presence of BEMD spores displayingendoglucanase or protease grew significantly taller than control B.t.spore treated or water control soybeans (statistical analysis assayedvia a t-test).

TABLE 5 Height, inches (cm) Comparison SEM H₂O 7.13 (18.11)   100% 0.721Bt Control 7.86 (19.96) 110.33% 0.752 BEMD Endo 9.75 (24.76) 136.80%0.21 BEMD Protease  8.8 (22.35) 123.40% 0.354

Example 4 Use of Recombinant Bacillus cereus Family Members Displayingan Endogluconase to Stimulate Plant Growth in Ryegrass

BEMD spores expressing endogluconase were created in an identicalfashion as described above in Example 1. Perennial ryegrass was planted0.25 inches (6.4 mm) deep in 10 cm deep pots filled with standard loamtopsoil. Spores, both control and BEMD expressing endogluconase, werediluted to a concentration of 1×10⁴/ml in 50 ml of water and applied toeach plant at planting. A water-only control was also included. Plantswere grown under ideal light using T5 lamps, 54 watts, and exposed to 11hours of light a day under controlled temperature conditions between60-78° F. (15.5-25.5° C.). Plants were watered to saturation every threedays over the two week trial. At the end of two weeks, the height ofeach plant was measured, and measurements were normalized to controlwater only plants.

Results are shown in Table 6, together with the standard error of themean. Ryegrass grown in the presence of BEMD spores displayingendocellulase grew significantly taller than control B.t. spore treatedor water control ryegrass (statistical analysis assayed via a t-test).

TABLE 6 Height, inches (cm) Comparison SEM H₂O  4.5 (11.43) 100.0% 0.137Bt Control 4.84 (12.29) 107.7% 0.128 BEMD Endo 5.03 (12.78) 111.9% 0.137

Example 5 Use of Recombinant Bacillus cereus Family Members DisplayingEnzymes Involved in the Synthesis of Plant Hormones to Stimulate PlantGrowth

The BEMD system can also be used to display enzymes involved in thesynthesis of plant hormones. For example, the plant hormoneindole-3-acetic acid is a potent growth stimulator in plants.Indole-3-acetic acid is synthesized in vivo from tryptophan by theenzymes tryptophan monoxygenase and indole-3-acetamide hydrolase.Indole-3-acetic acid and other auxin hormones can also be synthesized invivo from tryptophan and/or indole by the enzymes nitrilase, tryptophanaminotransferase, indole-3-acetaldehyde dehydrogenase, indole-3-pyruvatedecarboxylase, amine oxidase, tryptophan decarboxylase, and tryptophanside chain oxidases.

Related plant growth hormones (auxins) include indole-3-pyruvic acid,indole-3-acetaldoxime, indole-3-acetamide, indole-3-acetonitrile,indole-3-ethanol, indole-3-pyruvate, indole-3-butyric acid, phenylaceticacids, 4-chloroindole-3-acetic acid, and indole-3-acetaldoxime. Thesehormones are synthesized from tryptophan and/or indole in vivo via theenzymes tryptophan monoxygenase, indole-3-acetamide hydrolase,nitrilase, nitrile hydrolase, acetolactate synthetase, alphaacetolactate decarboxylase, tryptophan aminotransferase,indole-3-acetaldehyde dehydrogenase, indole-3-pyruvate decarboxylase,amine oxidase, tryptophan decarboxylase, and tryptophan side chainoxidases.

Growth hormones of the cytokinin family can also be synthesized byenzymes expressed in the BEMD system. Examples of cytokinins includekinetin, zeatin (cis and trans), 6-benzylaminopurine, dihydroxyzeatin,N6-(D2-isopentenyl)adenine, ribosylzeatin, N6-(D2-isopentenyl)adenosine,2 methylthio-cis-ribosylzeatin, cis ribosylzeatin,ribosylzeatin-5-monosphosphate, N6-methylaminopurine,N6-dimethylaminopurine, 2′-deoxyzeatin riboside,4-hydroxy-3-methyl-trans-2-butenylaminopurine, ortho-topolin,meta-topolin, benzyladenine, ortho-methyltopolin, andmeta-methyltopolin. These plant growth stimulating compounds aresynthesized in vivo from mevalonate or adenosine mono/di/triphosphate byenzymes including adenosine phosphate isopentenyltransferases,phosphatases, adenosine kinases, adenine phosphoribosyltransferase,CYP735A, 5′ ribonucleotide phosphohydrolase, adenosine nucleosidases,zeatin cis-trans isomerase, zeatin O-glucosyltransferases,β-glucosidases, cis-hydroxylases, CK cis-hydroxylases, CKN-glucosyltransferases, 2,5-ribonucleotide phosphohydrolases, adenosinenucleosidases, purine nucleoside phosphorylases, and zeatin reductases.

Using methods similar to those described above in Example 1, any ofthese enzymes can be incorporated into the BEMD system for display onBEMD spores by creating a fusion construct comprising the enzyme and atargeting sequence that targets the expressed enzyme to the exosporiumwhen the fusion construct is expressed in a Bacillus cereus familymember. A recombinant Bacillus cereus family member expressing such aconstruct can then be added to the soil or other plant growth medium orapplied directly to plant foliage using methods similar to thosedescribed above in Example 1 for stimulation of plant growth.

The plant growth medium can be supplemented with precursors orsubstrates for the enzymes. For example, the plant growth medium can besupplemented with tryptophan, adenosine monophosphates, adenosinediphosphates, adenosine triphosphates, or indole. Suitableconcentrations of these substrates are between 100 nM and 100 μM.

Example 6 Use of Recombinant Bacillus cereus Family Members DisplayingProteases or Peptidases that Cleave Proteins or Peptides into BioactivePeptides for Stimulation of Plant Growth

Proteases and peptidases can be expressed in the BEMD system that canenzymatically cleave available proteins in the plant growth media tobioactive peptides that can act on the plant directly or indirectly.Examples include the enzymatic cleavage of soybean meal, yeast extract,or other protein rich meals added to the plant growth medium into activepeptides that can directly stimulate plant growth. Bioactive peptidesgenerated by enzymatic cleavage of protein meals include RHPP and RKN16D10, potent stimulators of plant root development.

Using methods similar to those described above in Example 1, any ofthese proteases and peptidases can be incorporated into the BEMD systemfor display on BEMD spores by creating a fusion construct comprising theprotease or peptidase and a targeting sequence that targets theexpressed enzyme to the exosporium when the fusion construct isexpressed in a Bacillus cereus family member. A recombinant Bacilluscereus family member expressing such a construct can then be added tosoil or other plant growth medium supplemented with soybean meal, yeastextract, or another-protein-rich meal for stimulation of plant growth.The soybean meal, yeast extract, or other protein-rich meal is suitablyadded to the plant growth medium in the form of a liquid compositioncomprising about 10 μg/L to about 100 mg/L of the protein meal, yeastextract, or other protein-rich meal.

Example 7 Use of Recombinant Bacillus cereus Family Members DisplayingProteins or Peptides Involved in the Stimulation of Plant Growth

The BEMD system can also be used to display proteins or peptides thatare directly involved in the promotion of plant growth. For example,plant peptide hormones or non-hormone peptides that stimulate plantgrowth can be expressed in the BEMD system. For example, non-hormonepeptides that directly bind to and active plant receptors can beexpressed in the BEMD system to directly act on receptors in the plantand roots of target plants. Such peptide hormones and bioactive peptidesinclude phytosulfokine, calcalva 3 (CLV3), systemin, RKN 16D10,Hg-Syv46, eNOD40, ZmlGF, SCR/SP11 family proteins and peptides, RHPP,and KTI (kunitz trypsin inhibitor). These peptides and related peptidescan be expressed in the BEMD system and delivered to plant growth mediumor directly applied to foliage to stimulate plant growth.

Using methods similar to those described above in Example 1, any ofthese proteins or peptides can be incorporated into the BEMD system fordisplay on BEMD spores by creating a fusion construct comprising theenzyme and a targeting sequence that targets the expressed enzyme to theexosporium when the fusion construct is expressed in a Bacillus cereusfamily member. A recombinant Bacillus cereus family member expressingsuch a construct can then be added to the soil or other plant growthmedium or applied directly to plant foliage using methods similar tothose described above in Example 1 for stimulation of plant growth.

Example 8 Use of Recombinant Bacillus cereus Family Members DisplayingEnzymes that Degrade or Modify a Bacterial, Fungal, or Plant NutrientSource to Stimulate Plant Growth

The BEMD system can also be used display enzymes that degrade or modifybeneficially a bacterial, fungal, or plant nutrient source present insoil or another plant growth medium. Such enzymes degrade productspresent in the soil or other plant growth medium into forms that caneasily be taken up by plants and/or the beneficial bacteria and/or fungiof the rhizosphere. Such enzymes include, for example, glucosidehydrolases to degrade complex carbohydrates, cellulases to degradecellulose; lipases to degrade lipids, including oil, fats, and waxes;lignin oxidases to degrade down lignin and humic acids; and proteases todegrade polypeptides. The resultant products, including simple sugars,amino acids, fatty acids, and other nutrients will be readily availablefor direct uptake by plants and/or for stimulating beneficial bacteriaand/or fungi to grow and thrive in the rhizospheres of the plants.

In addition, enzymes and other biological molecules can be utilized torelease or sequester phosphate, nitrogen, and other key elementalnutrients for plant uptake from their various organic and inorganicforms in soil. For example, phosphatases can be used to degradephosphates in the environment into usable inorganic phosphates for plantuse. The phosphates can be naturally occurring phosphates present in aplant growth medium. Alternatively, the plant growth medium can besupplemented with phosphates such as trimetaphosphate, a commonagricultural amendment. Examples of useful phosphatases includephosphoric monoester hydrolases, phosphomonoesterases, phosphoricdiester hydrolases, phosphodiesterases, triphosphoric monoesterhydrolases, phosphoryl anhydride hydrolases, pyrophosphatases, phytase,trimetaphosphatases, and triphosphatases. For example, the enzymestrimetaphosphatase, triphosphatase, and pyrophosphatase sequentiallybreak down trimetaphosphate into usable inorganic phosphate.

The nitrogenase family of enzymes convert atmospheric nitrogen (N₂) intoammonia, thereby converting nitrogen that would otherwise beunaccessible to plants into a usable form. Suitable enzymes belong tothe Nif family of nitrogenases.

Chemical energy can also be directly added into the plant growth mediumas adenosine-3-triphosphate, ferrodoxin, or additional enzymes thatcreate such energy into the BEMD system. These are cofactors for thenitrogenases and are limited in soil. Thus, such cofactors can be addedto soil to enhance the reactions described above.

Using methods similar to those described above in Example 1, any ofthese enzymes can be incorporated into the BEMD system for display onBEMD spores by creating a fusion construct comprising the enzyme and atargeting sequence for targeting the fusion construct to the exosporiumof a Bacillus cereus family member. The fusion construct can then beexpressed in a Bacillus cereus family member, and this recombinantBacillus cereus family member can be added to soil or another plantgrowth medium using methods similar to those described above in Example1 for stimulation of plant growth.

Example 9 Use of Recombinant Bacillus cereus Family Members DisplayingEnzymes Involved in the Synthesis of 2,3-Butanediol for Stimulation ofPlant Growth

The BEMD system can also be used display enzymes involved in thesynthesis of the plant growth promoting compound 2,3-butanediol. Invivo, 2,3-butanediol is synthesized by beneficial bacteria and fungi inthe rhizosphere from acetoin, diacetyl, acetolactate, or pyruvate by theenzymes acetolactate synthetase, α-acetolactate decarboxylase, pyruvatedecarboxylase, diacetyl reductase, butanediol dehydrogenases, andacetoin reductase.

Any of these enzymes can be incorporated into the BEMD system fordisplay on BEMD spores using methods similar to those described above inExample 1. A fusion construct can be prepared that comprises the enzymeand a targeting sequence that targets the enzyme to the exosporium whenthe fusion construct is expressed in a Bacillus cereus family member.The fusion construct is then expressed in a Bacillus cereus familymember, and the Bacillus cereus family member is added to soil oranother plant growth medium for stimulation of plant growth.

To increase the effect of the enzymes displayed on BEMD, the soil can besupplemented with substrates for the enzymes. For example, the soil canbe supplemented with acetoin, which is a substrate for acetoinreductase; pyruvate, which is a substrate for pyruvate decarboxylase;diacetyl, which is a substrate for diacetyl reductase; and/oracetolactate, which is a substrate for acetolactate decarboxylase.

Example 10 Use of Recombinant Bacillus cereus Family Members DisplayingProteases for Protecting Plants from Pathogens

The BEMD system can also be used display proteases that protect plantsfrom one or more pathogens. For example, certain bacterial pathogens cancommunicate between individual members via secretion of bacteriallactone homoserines or related signaling molecules. Thus, proteasesspecific for bacterial lactone homoserine signaling molecules canprotect plants from such bacterial pathogens by disrupting communicationbetween bacteria, a step essential for the bacteria to secrete toxinsand upregulate virulence factors. Suitable proteases specific forbacterial lactone homoserine signaling molecules include endopeptidasesand exopeptidases.

Proteases specific for bacterial lactone homoserine signaling moleculescan be incorporated into the BEMD system using methods similar to thosedescribed above in Example 1. A fusion construct can be prepared thatcomprises the protease and a targeting sequence that targets theprotease to the exosporium when the fusion construct is expressed in aBacillus cereus family member. The fusion construct is then expressed ina Bacillus cereus family member, and the Bacillus cereus family memberis added to soil or another plant growth medium. The protease can thendegrade the bacterial lactone homoserine signaling molecules, blocking akey step in the virulence of these organisms and thereby helping toprotect the plant from these pathogens.

Example 11 Use of Recombinant Bacillus cereus Family Members DisplayingAntimicrobial Proteins and Peptides for Protecting Plants from Pathogens

The BEMD system can also be used display enzymes that exhibitantibacterial and/or antifungal activities that can help protect plantsfrom one or more pathogens. For example, antibacterial proteins such asbacteriocins, lysozymes, siderophores, avidins, streptavidins,conalbumin, albumin, and lactoferrin can all be expressed in the BEMDsystem to exert their effect on bacterial and fungal pathogens ofplants. Bacteriocins, albumin, conalbumin, lysozymes, and lactoferrinexert direct antimicrobial action on their targets, whereassiderophores, avidins, streptavidins bind essential nutrients thatpathogens require for virulence. For example, the peptide lactoferrin,when expressed on the surface of the BEMD system would lyse bacteriacells that are susceptible to the lactoferrin peptides in the plantgrowth medium. These proteins and peptides have specific action onselect microbes, and can selectively target a group of pathogens withoutobstructing all microbes in the plant growth medium.

Any of these proteins or peptides can be incorporated into the BEMDsystem for display on BEMD spores using methods similar to thosedescribed above in Example 1. A fusion construct can be prepared thatcomprises the enzyme and a targeting sequence that targets the enzyme tothe exosporium when the fusion construct is expressed in a Bacilluscereus family member. The fusion construct is then expressed in aBacillus cereus family member, and the Bacillus cereus family member isadded to soil or another plant growth medium for protection of plantsfrom one or more pathogens.

Example 12 Use of Recombinant Bacillus cereus Family Members DisplayingEnzymes for Protecting Plants from Pathogens

The BEMD system can also be used display enzymes that protect plantsfrom one or more pathogens. For example, yeast and mold cell walls aredegraded by enzymes such as β-1,3-glucanases, β-1,4-glucanases,β-1,6-glucanases, chitosinases, chitinases, chitosinase-like proteins,and lyticases. Bacteria cell walls are degraded by enzymes selected fromproteinases, proteases, mutanolysin, stapholysin, and lysozymes. Each ofthese cell wall degrading enzymes can be expressed on the BEMD systemand added to plant growth medium for selective inhibition of pathogenicmicrobes in the rhizosphere.

Any of these proteins or peptides can be incorporated into the BEMDsystem for display on BEMD spores using methods similar to thosedescribed above in Example 1. A fusion construct can be prepared thatcomprises the enzyme and a targeting sequence that targets the enzyme tothe exosporium when the fusion construct is expressed in a Bacilluscereus family member. The fusion construct is then expressed in aBacillus cereus family member, and the Bacillus cereus family member isadded to soil or another plant growth medium for protection of plantsfrom pathogens.

Example 13 Use of Recombinant Bacillus cereus Family Members DisplayingPlant Immune System Stimulatory Peptides or Proteins for ProtectingPlants from Pathogens

The BEMD system can also be used display plant immune system enhancerpeptides and proteins. These proteins can be expressed on the outside ofthe BEMD spore and delivered into the plant growth medium to stimulatethe plant immune system to allow the plant to protect itself from plantpathogens. Example proteins and peptides include harpin, α-elastins,β-elastins, cryptogein, and flagellin proteins and peptides. Exposure ofplants to these proteins and peptides will stimulate resistance to manyplant pathogens in plants.

Any of these proteins or peptides can be incorporated into the BEMDsystem for display on BEMD spores using methods similar to thosedescribed above in Example 1. A fusion construct can be prepared thatcomprises the enzyme and a targeting sequence that targets the enzyme tothe exosporium when the fusion construct is expressed in a Bacilluscereus family member. The fusion construct is then expressed in aBacillus cereus family member, and the Bacillus cereus family member isadded to soil or another plant growth medium for protection of plantsfrom pathogens.

Example 14 Use of Recombinant Bacillus cereus Family Members Displayinga Root Binding Protein or Peptide to Immobilize the Recombinant Bacilluscereus Family Member on a Root System of a Plant

Root binding proteins and peptides can also be incorporated into theBEMD system to allow the BEMD spores to be immobilized on a root systemof a plant. Display of such root binding ligands on the BEMD sporesallows for targeting of the spores to the root system of a plant or tosubstructures of the root system to maintain the BEMD spores at anoptimal location for other displayed biological molecules and enzymes tobe effective.

For example, rhicadhesin is a root binding ligand that binds to roothairs. Thus, display of rhicadhesin on the BEMD spores thus targets thespores to root hairs. Additional proteins that could be utilized forselective binding to plants include adhesins, falgellin, omptins,lectins, pili proteins, curlus proteins, intimins, invasins, agglutinin,and afimbrial proteins.

Such root binding proteins and peptides can be incorporated into theBEMD system using methods similar to those described above in Example 1.A fusion construct can be prepared that comprises the root bindingprotein or peptide and a targeting sequence that targets the protein orpeptide to the exosporium when the construct is expressed in a Bacilluscereus family member. The fusion construct containing the root bindingligand is then expressed in a Bacillus cereus family member. Such fusionconstructs can be coexpressed with one or more additional fusionconstructs comprising any of the beneficial enzymes discussed herein(e.g., an enzyme involved in the synthesis of a plant hormone, an enzymethat degrades a nutrient source, or a proteases that protects a plantfrom a pathogen). The recombinant Bacillus cereus family member is addedto soil or another plant growth medium. The root binding ligand targetsthe Bacillus cereus family member to the root system of the plant andimmobilizes it there, thus allowing the coexpressed fusion construct toexert its effects in close proximity to the root system.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above fusion proteins, Bacilluscereus family members, formulations, and methods without departing fromthe scope of the invention, it is intended that all matter contained inthe above description and shown in the accompanying drawing shall beinterpreted as illustrative and not in a limiting sense.

What is claimed is:
 1. A method for stimulating plant growth comprising:applying a recombinant Bacillus cereus family member expressing a fusionprotein to a plant seed, wherein applying the recombinant Bacilluscereus family member to the plant seed comprises: (a) applying therecombinant Bacillus cereus family member to the plant seed at the timeof planting; or (b) coating the plant seed with a seed coatingformulation comprising the recombinant Bacillus cereus family member andan agriculturally acceptable carrier; and contacting the seed with aplant growth medium; wherein the fusion protein comprises at least oneplant growth stimulating protein or peptide and a targeting sequence orexosporium protein that targets the fusion protein to the exosporium ofthe recombinant Bacillus cereus family member, and wherein plants grownfrom plant seeds to which the recombinant Bacillus cereus family memberhas been applied exhibit increased growth as compared to plants grownfrom plant seeds to which the recombinant Bacillus cereus family memberhas not been applied, under the same conditions.
 2. A method of claim 1,wherein the fusion protein comprises a targeting sequence comprising anamino acid sequence having at least about 43% identity with amino acids20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is atleast about 54%.
 3. A method of claim 2, wherein the targeting sequencecomprises an amino acid sequence having at least about 50% identity withamino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids25-35 is at least about 63%.
 4. A method of claim 2, wherein thetargeting sequence comprises an amino sequence having at least about 62%identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identitywith amino acids 25-35 is at least about 72%.
 5. A method of claim 2,wherein the targeting sequence comprises an amino acid sequence havingat least about 81% identity with amino acids 20-35 of SEQ ID NO: 1,wherein the identity with amino acids 25-35 is at least about 90%.
 6. Amethod of claim 2, wherein the targeting sequence consists of: (a) a 16amino acid sequence having at least about 43% identity with amino acids20-35 of SEQ ID NO. 1, wherein the identity with amino acids 25-35 is atleast about 54%; (b) amino acids 1-35 of SEQ ID NO. 1; (c) amino acids20-35 of SEQ ID NO. 1; or (d) SEQ ID NO.
 1. 7. A method of claim 1,wherein the targeting sequence comprises the amino acid sequence GXT atits carboxy terminus, wherein X is any amino acid.
 8. A method of claim1, wherein the plant growth stimulating protein comprises an enzyme. 9.A method of claim 8, wherein the enzyme comprises a cellulase.
 10. Amethod of claim 9, wherein the cellulase comprises an endocellulase, anexocellulase, or a β-glucosidase.
 11. A method of claim 10, wherein theendocellulase comprises a Bacillus subtilis endoglucanase, a Bacillusthuringiensis endoglucanase, a Bacillus cereus endoglucanase, or aBacillus clausii endoglucanase.
 12. A method of claim 8, wherein theenzyme comprises a lipase.
 13. A method of claim 12, wherein the lipasecomprises a Bacillus subtilis lipase, a Bacillus thuringiensis lipase, aBacillus cereus lipase, or a Bacillus clausii lipase.
 14. A method ofclaim 8, wherein the enzyme comprises a lignin oxidase.
 15. A method ofclaim 14, wherein the lignin oxidase comprises a lignin peroxidase, alaccase, a glyoxal oxidase, a ligninase, or a manganese peroxidase. 16.A method of claim 8, wherein the enzyme comprises a protease.
 17. Amethod of claim 16, wherein the protease comprises a subtilisin, an acidprotease, an alkaline protease, a proteinase, a peptidase, anendopeptidase, an exopeptidase, a thermolysin, a papain, a pepsin, atrypsin, a pronase, a carboxylase, a serine protease, a glutamicprotease, an aspartate protease, a cysteine protease, a threonineprotease, or a metalloprotease.
 18. A method of claim 8, wherein theenzyme comprises a glycoside hydrolase.
 19. A method of claim 8, whereinthe enzyme comprises a phosphatase.
 20. A method of claim 19, whereinthe phosphatase comprises a phosphoric monoester hydrolase, aphosphomonoesterase, a phosphoric diester hydrolase, aphosphodiesterase, a triphosphoric monoester hydrolase, a phosphorylanhydride hydrolase, a pyrophosphatase, a phytase, a trimetaphosphatase,or a triphosphatase.
 21. A method of claim 8, wherein the enzymecomprises a nitrogenase.
 22. A method of claim 21, wherein thenitrogenase comprises a Nif family nitrogenase.
 23. A method of claim 8,wherein the enzyme comprises a nuclease.
 24. A method of claim 8,wherein the plant growth medium is supplemented with a substrate for anenzyme.
 25. A method of claim 24, wherein the substrate comprisestryptophan, an adenosine monophosphate, an adenosine diphosphate, anadenosine triphosphate, indole, a trimetaphosphate, ferrodoxin, acetoin,diacetyl, pyruvate, acetolactate, or a combination thereof.
 26. A methodof claim 1, comprising coating the plant seed with the seed coatingformulation.
 27. A method of claim 1, wherein the recombinant Bacilluscereus family member comprises Bacillus anthracis, Bacillus cereus,Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides,Bacillus weihenstephensis, or a combination thereof.
 28. A method ofclaim 1, wherein the plant growth stimulating protein or peptidecomprises a peptide hormone.
 29. A method of claim 28, wherein thepeptide hormone comprises a phytosulfokine, clavata 3 (CLV3), systemin,ZmlGF, or a SCR/SP11.
 30. A method of claim 1, wherein the plant growthstimulating protein or peptide comprises a non-hormone peptide.
 31. Amethod of claim 30, wherein the non-hormone peptide comprises a RKN16D10, Hg-Syv46, an eNOD40 peptide, RHPP, or kunitz trypsin inhibitor.32. A method of claim 1, wherein the plant growth stimulating protein orpeptide is physically attached to the exosporium of the recombinantBacillus cereus family member.
 33. A method of claim 1, furthercomprising inactivating the recombinant Bacillus cereus family memberprior to application to the plant seed.
 34. A method of claim 1, whereinthe fusion protein comprises: (a) a targeting sequence comprising aminoacids 1-35 of SEQ ID NO: 1; (b) a targeting sequence comprising aminoacids 20-35 of SEQ ID NO: 1; (c) a targeting sequence comprising SEQ IDNO: 1; or (d) an exosporium protein comprising SEQ ID NO:
 2. 35. Amethod of claim 1, wherein the fusion protein comprises: (a) a targetingsequence comprising amino acids 1-27 of SEQ ID NO: 3; (b) a targetingsequence comprising amino acids 12-27 of SEQ ID NO: 3; (c) a targetingsequence comprising SEQ ID NO: 3; or (d) an exosporium proteincomprising SEQ ID NO:
 4. 36. A method of claim 1, wherein the fusionprotein comprises: (a) a targeting sequence comprising amino acids 1-38of SEQ ID NO: 5; (b) a targeting sequence comprising amino acids 23-38of SEQ ID NO: 5; (c) a targeting sequence comprising SEQ ID NO: 5; or(d) an exosporium protein comprising SEQ ID NO:
 6. 37. A method of claim1, wherein the fusion protein comprises: (a) a targeting sequencecomprising amino acids 1-28 of SEQ ID NO: 7; (b) a targeting sequencecomprising amino acids 13-28 of SEQ ID NO: 7; (c) a targeting sequencecomprising SEQ ID NO: 7; or (d) an exosporium protein comprising SEQ IDNO:
 8. 38. A method of claim 1, wherein the fusion protein comprises:(a) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 9;(b) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 9;(c) a targeting sequence comprising SEQ ID NO: 9; or (d) an exosporiumprotein comprising SEQ ID NO:
 10. 39. A method of claim 1, wherein thefusion protein comprises: (a) a targeting sequence comprising aminoacids 1-33 of SEQ ID NO: 11; (b) a targeting sequence comprising aminoacids 18-33 of SEQ ID NO: 11; (c) a targeting sequence comprising SEQ IDNO: 11; or (c) an exosporium protein comprising SEQ ID NO:
 12. 40. Amethod of claim 1, wherein the fusion protein comprises: (a) a targetingsequence comprising amino acids 1-33 of SEQ ID NO: 13; (b) a targetingsequence comprising amino acids 18-33 of SEQ ID NO: 13; (c) a targetingsequence comprising amino SEQ ID NO: 13; or (d) an exosporium proteincomprising SEQ ID NO:
 14. 41. A method of claim 1, wherein the fusionprotein comprises: (a) a targeting sequence comprising amino acids 1-43of SEQ ID NO: 15; (b) a targeting sequence comprising amino acids 28-43of SEQ ID NO: 15; (c) a targeting sequence comprising amino SEQ ID NO:15; or (d) an exosporium protein comprising SEQ ID NO:
 16. 42. A methodof claim 1, wherein the fusion protein comprises: (a) a targetingsequence comprising amino acids 1-27 of SEQ ID NO: 17; (b) a targetingsequence comprising amino acids 12-27 of SEQ ID NO: 17; (c) a targetingsequence comprising amino SEQ ID NO: 17; or (d) an exosporium proteincomprising SEQ ID NO:
 18. 43. A method of claim 1, wherein the fusionprotein comprises: (a) a targeting sequence comprising amino acids 1-33of SEQ ID NO: 19; (b) a targeting sequence comprising amino acids 18-33of SEQ ID NO: 19; (c) a targeting sequence comprising amino SEQ ID NO:19; or (d) an exosporium protein comprising SEQ ID NO:
 20. 44. A methodof claim 1, wherein the fusion protein comprises: (a) a targetingsequence comprising amino acids 1-33 of SEQ ID NO: 21; (b) a targetingsequence comprising amino acids 18-33 of SEQ ID NO: 21; (c) a targetingsequence comprising amino SEQ ID NO: 21; or (d) an exosporium proteincomprising SEQ ID NO:
 22. 45. A method of claim 1, wherein the fusionprotein comprises: (a) a targeting sequence comprising amino acids 1-24of SEQ ID NO: 23; (b) a targeting sequence comprising amino acids 9-24of SEQ ID NO: 23; (c) a targeting sequence comprising amino SEQ ID NO:23; or (d) an exosporium protein comprising SEQ ID NO:
 24. 46. A methodof claim 1, wherein the fusion protein comprises: (a) a targetingsequence comprising amino acids 1-24 of SEQ ID NO: 25; (b) a targetingsequence comprising amino acids 9-24 of SEQ ID NO: 25; (c) a targetingsequence comprising amino SEQ ID NO: 25; or (d) an exosporium proteincomprising SEQ ID NO:
 26. 47. A method of claim 1, wherein the fusionprotein comprises: (a) a targeting sequence comprising amino acids 1-30of SEQ ID NO: 27; (b) a targeting sequence comprising amino acids 15-30of SEQ ID NO: 27; (c) a targeting sequence comprising amino SEQ ID NO:27; or (d) an exosporium protein comprising SEQ ID NO:
 28. 48. A methodof claim 1, wherein the fusion protein comprises: (a) a targetingsequence comprising amino acids 1-33 of SEQ ID NO: 29; (b) a targetingsequence comprising amino acids 18-33 of SEQ ID NO: 29; (c) a targetingsequence comprising amino SEQ ID NO: 29; or (d) an exosporium proteincomprising SEQ ID NO:
 30. 49. A method of claim 1, wherein the fusionprotein comprises: (a) a targeting sequence comprising amino acids 1-24of SEQ ID NO: 31; (b) a targeting sequence comprising amino acids 9-24of SEQ ID NO: 31; (c) a targeting sequence comprising amino SEQ ID NO:31; or (d) an exosporium protein comprising SEQ ID NO:
 32. 50. A methodof claim 1, wherein the fusion protein comprises: (a) a targetingsequence comprising amino acids 1-15 of SEQ ID NO: 33; (b) a targetingsequence comprising amino SEQ ID NO: 33; or (c) an exosporium proteincomprising SEQ ID NO:
 34. 51. A method of claim 1, wherein the fusionprotein comprises: (a) a targeting sequence comprising amino acids 1-16of SEQ ID NO: 35; (b) a targeting sequence comprising amino SEQ ID NO:35; or (c) an exosporium protein comprising SEQ ID NO: 36.